Pregabalin immunoassays

ABSTRACT

Compounds and methods for use in detecting pregabalin in a sample suspected of containing pregabalin are disclosed. Pregabalin derivatives are described for producing pregabalin conjugates. A pregabalin-immunogenic carrier conjugate may be used as an immunogen for the preparation of an anti-pregabalin antibody. A pregabalin-detectable label conjugate may be used in a signal producing system in pregabalin assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/936,158, filed Mar. 26, 2018, now U.S. Pat. No. 10,690,687, which isa divisional of U.S. application Ser. No. 15/045,969, filed Feb. 17,2016, now U.S. Pat. No. 9,958,464, which claims priority pursuant to 35U.S.C. § 119 (e) to the filing date of U.S. Provisional Application Ser.No. 62/127,689 filed on Mar. 3, 2015, the disclosures of each of whichare herein incorporated by reference.

INTRODUCTION

Pregabalin is an analogue of the neurotransmitter gamma amino butyricacid (GABA) with analgesic, anticonsulvant and anxiolytic activity.Pregabalin is described chemically as(S)-3-(aminomethyl)-5-methylhexanoic acid, and its structure is shownbelow and in FIG. 1 , panel A.

Pregabalin is indicated for neuropathic pain associated with diabeticperipheral neuropathy, postherpetic neuralgia, adjunctive therapy foradult patients with partial onset seizures, fibromyalgia, neuropathicpain associated with spinal cord injury (see, e.g., LYRICA®). In Europe,pregabalin is also approved to treat generalized anxiety disorder.

Recent reports have suggested that pregabalin is used as a recreationaldrug and that an abuse potential may exist. Pregabalin is classified asa schedule V drug in the U.S. Drug Enforcement Administration'sControlled Substances Act. In the European Union, Pregabalin is not acontrolled substance subjected to special or restricted prescription,but a warning related to its abuse potential was added to the Summary ofProduct Characteristics in June 2010. Based upon the potential forillicit use of Pregabalin as a recreational drug, urinary drug testingmay be a tool to identify its use.

In addition, therapeutic drug management (TDM) of pregabalin can serveas a tool to ensure compliance in administering chemotherapy with theactual prescribed dosage and achievement of effective serumconcentration levels. TDM can provide the clinician with insight onpatient variation, and allow the clinical to individualize drug dosagesto the patient's needs. Successful pain management may be facilitated byconsistent monitoring to ensure that the dosage is taken as prescribed.

A variety of analytical methods have been developed for the measurementof pregabalin in blood and urine including gas chromatography coupledwith mass spectrometry (GC-MS) and (ultra-) high-performance liquidchromatography coupled with mass spectrometry ((U)HPLC-MS) orfluorometric detection and capillary electrophoresis coupled totime-of-flight mass spectrometry detection (CE/TOF/MS). Whilechromatographic techniques can be used to determine drug levels, suchmethods are impractical for commercial use due to, for example, longsample preparation time, long assay time, high cost, and labor-intensiveprocedures. Immunoassays provide simple and fast analytical methods fordetection and measurement of drug levels.

SUMMARY

Methods, compositions and kits are disclosed directed at pregabalinderivatives, immunogens, signal generating moieties, antibodies thatbind pregabalin and immunoassays for detection of pregabalin.

Aspects of the present disclosure include a compound of Formula (A):

wherein

-   -   R¹ or R² is —X—W-L-Z, wherein        -   when R¹ is —X—W-L-Z, then R² is —NH₂,        -   when R² is —X—W-L-Z, then R is —OH, and    -   X is NH;    -   W is selected from a bond, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,        substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl, and        carbonyl;    -   L is a bond or a linker; and    -   Z is selected from H, alkyl, substituted alkyl, a reactive        functional group, an immunogenic carrier and a detectable label;    -   or a salt thereof.

In some embodiments, W is a bond. In some embodiments, W is an alkyl orsubstituted alkyl.

In some embodiments, W is a carbonyl.

In some embodiments, the linking group includes 1-15 carbon atoms and/or0-6 heteroatoms.

In some embodiments, the linker is selected from:

—(CH₂)_(n)C(O)—,

—C(O)(CH₂)_(n)—,

—C(O)(CH₂)_(n)NH—C(O)—,

—C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,

—(CH₂)_(n)SCH₂C(O)—,

—(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,

—(CH₂)_(m)C(O)NH(CH₂)_(n)—,

—(CH₂)_(n)NH—C(O)—,

—(CH₂)_(m)NH—C(O)(CH₂)_(n)—,

—C(O)—(CH₂)_(n)—, and

—(CH₂)_(n)—;

wherein m and n are each independently selected from an integer from 0to 10.

In some embodiments, Z is an immunogenic carrier. In some embodiments,the immunogenic carrier is a protein. In some embodiments, the proteinis selected from the group consisting of hemocyanins, globulins andalbumins. In some embodiments, the protein is bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). In some embodiments, the immunogeniccarrier is a polysaccharide. In some embodiments, Z is a detectablelabel. In some embodiments, the detectable label is an enzyme. In someembodiments, the enzyme is selected from glucose-6-phosphatedehydrogenase (G6PDH), alkaline phosphatase, β-galactosidase, and horseradish peroxidase. In some embodiments, the enzyme isglucose-6-phosphate dehydrogenase (G6PDH).

In some embodiments, Z is a reactive functional groups selected fromhalogen, —OH, —SH, —NH₂, —O-lower alkyl, epoxy, —S-acyl, carboxyl,maleimidyl, haloacetamide, hydroxysuccinimidyl, succinimidyl, carbonate,anhydride, imidate, an isocyanate, an isothiocyanate, an imidoester, amaleimide, a thiolactone, a diazonium group, an acrylamide, an acylazide, an acyl nitrile, an alkyl halide, an aniline, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a halotriazine, ahydrazine, a hydrazide, an imido ester, a phosphoramidite, a reactiveplatinum complex, a sulfonyl halide, and a photoactivatable group

Aspects of the present disclosure include a method for detectingpregabalin. In some embodiments, the method includes: combining in areaction mixture a sample suspected of containing pregabalin with anantibody that binds pregabalin or a conjugate that includes a compoundof the Formula (A) of claim 1; and detecting the presence or absence ofa complex that includes the pregabalin and the antibody, where thepresence of the complex indicates the presence of pregabalin in thesample.

In some embodiments, the antibody is a polyclonal antibody. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody has a cross-reactivity with crossreactants of 0.1% or lesswith respect to binding to pregabalin.

In some embodiments, the reaction mixture includes the conjugate. Insome embodiments, the conjugate includes a detectable label.

In some embodiments, the detecting includes determining the presence ofan enzymatic reaction product of the conjugate.

Aspects of the present disclosure include an isolated antibody thatbinds an epitope present in pregabalin and present in a compound ofFormula (A) of the present disclosure.

In some embodiments, the antibody is a polyclonal antibody. In someembodiments, the antibody is a monoclonal antibody.

Aspects of the present disclosure include a kit for detecting pregabalinin a sample. The kit includes: an antibody as described herein; andinstructions for an assay for detecting pregabalin.

In some embodiments, the kit includes one or more reagents for detectinga complex of the antibody and pregabalin. In some embodiments, the kitincludes a compound of Formula (A) of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows chemical structures of pregabalin[(S)-3-(aminomethyl)-5-methylhexanoic acid](FIG. 1 , panel A);gabapentin [2-[1-(aminomethyl)cyclohexyl]acetic acid)] (FIG. 1 , panelB); L-leucine (FIG. 1 , panel C); and a fragment of pregabalin with the3-isobutyl group, (S)-3-(aminomethyl) group, and carbon-3 of pregabalinidentified (FIG. 1 , panel D).

FIG. 2 is a schematic showing the structures of pregabalin derivatives,Hapten-A and Hapten-B, according to embodiments of the presentdisclosure.

FIG. 3 is a schematic representation of the reparation of KLH-SH,according to embodiments of the present disclosure.

FIG. 4 is a schematic representation of preparation of wild typeG6PDH-SH, according to embodiments of the present disclosure.

FIG. 5 is a schematic of the structures of a pregabalin conjugateincluding KLH-A, where pregabalin derivative Hapten-A is conjugated withKLH-SH, according to embodiments of the present disclosure.

FIG. 6 is a schematic of the structures of a pregabalin conjugateincluding KLH-B, where pregabalin derivative Hapten-B is conjugated withKLH-SH, according to embodiments of the present disclosure.

FIG. 7 is a schematic representation of rG6PDH and the structures of apregabalin conjugate including rG6PDH-A, where pregabalin derivativeHapten-A is conjugated with rG6PDH, according to embodiments of thepresent disclosure.

FIG. 8 is a schematic representation of native G6PDH and the structuresof a pregabalin conjugate including G6PDH-B, where pregabalin derivativeHapten-B is conjugated with G6PDH, according to embodiments of thepresent disclosure.

FIG. 9 is a schematic representation of rG6PDH and the structures of apregabalin conjugate including recombinant rG6PDH-B, where pregabalinderivative Hapten-B is conjugated with rG6PDH, according to embodimentsof the present disclosure.

FIG. 10 , panel 1, and FIG. 10 , panel 2, show schematic representationsof a homogeneous, competitive immunoassay for pregabalin using ananti-pregabalin antibody and a pregabalin-G6PDH enzyme conjugateaccording to embodiments of the present disclosure. As shown in reactionscheme 1 in FIG. 10 , panel 1, in the absence of plasma drug(pregabalin), the anti-pregabalin antibody binds to thepregabalin-enzyme (G6PDH) conjugate and inactivates the enzyme. As shownin reaction scheme 2 in FIG. 10 , panel 2, in the presence of plasmadrug, pregabalin from plasma, if present, competes with thepregabalin-G6PDH conjugate for binding to the antibody, thus allowingsome fraction of the pregabalin-G6PDH conjugate to become active andconvert NAD⁺ to NADH.

FIG. 11 is a calibration curve showing the change in optical densityaccording to the concentration of pregabalin in a sample, according toembodiments of the present disclosure.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the present disclosure.

An “analyte” is the compound or composition to be measured, the materialof interest, such as pregabalin. In certain embodiments, the analyte isa member of a specific binding pair (sbp) and may be a ligand, which ismono- or polyvalent, may be antigenic or haptenic, and is a singlecompound or plurality of compounds which share at least one commonepitopic or determinant site.

Any sample which is reasonably suspected of containing analyte can beanalyzed by the methods of the present disclosure. Such samples caninclude human, animal or man-made samples. The sample can be prepared inany convenient medium which does not interfere with the assay. Thesample may be an aqueous solution or a natural fluid, such as, urine,whole blood, serum, plasma, cerebral-spinal fluid, or saliva. In someembodiments, the sample is serum.

“Human serum”, as used herein, refers to the aqueous portion of humanblood remaining after the fibrin and suspended material (such as cells)have been removed.

A “pregabalin derivative” as used in this disclosure refers to acompound sharing a core structure with pregabalin and that can competewith pregabalin for binding to an anti-pregabalin binding partner, suchas an anti-pregabalin antibody.

Certain compounds disclosed herein in connection with embodiments of thepresent disclosure can exist in unsolvated forms as well as solvatedforms, including hydrated forms. In general, the solvated forms areequivalent to unsolvated forms and are encompassed within the referenceto the compounds set out in the present disclosure. Certain compoundsdisclosed herein in connection with embodiments of the presentdisclosure may exist in multiple crystalline or amorphous forms.

Certain compounds disclosed herein in connection with embodiments of thepresent disclosure possess asymmetric carbon atoms (optical centers) ordouble bonds; the racemates, diastereomers, geometric isomers andindividual isomers are encompassed within the scope of the presentdisclosure.

The compounds may be prepared as a single isomer (e.g., enantiomer,cis-trans, positional, diastereomer) or as a mixture of isomers. Incertain embodiments, the compounds are prepared as substantially asingle isomer. Methods of preparing substantially isomerically purecompounds are known in the art. For example, enantiomerically enrichedmixtures and pure enantiomeric compounds can be prepared by usingsynthetic intermediates that are enantiomerically pure in combinationwith reactions that either leave the stereochemistry at a chiral centerunchanged or result in its complete inversion. Alternatively, the finalproduct or intermediates along the synthetic route can be resolved intoa single stereoisomer. Techniques for inverting or leaving unchanged aparticular stereocenter, and those for resolving mixtures ofstereoisomers are well known in the art and it is well within theability of one of ordinary skill in the art to choose an appropriatemethod for a particular situation. See, generally, Furniss et al.(eds.), Vogel's Encyclopedia of Practical Organic Chemistry, 5th ed.,Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816; andHeller, Acc. Chem. Res. 23: 128 (1990).

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—. Use of a single dash (“-”) or double dash (“−” or “--”) refersto a single covalent bond, while use of “=” refers to a double bond. Thesymbol, )₂ or ₂ (when displayed with —S, indicates that the compoundinside the parenthesis may be present as a dimer forming a disulfidebond. The dimer may be reduced to a monomer.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof, havingthe stated number of carbon atoms and an acyl radical on at least oneterminus of the alkane radical. The “acyl radical” is the group derivedfrom a carboxylic acid by removing the —OH moiety therefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e., C₁-C₁₀ means one to ten carbons). Examples ofsaturated hydrocarbon radicals include, but are not limited to, groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.”, where “heteroalkyl” refers to carbon chains having oneor more substitutions at one or more carbon atoms of the hydrocarbonchain fragment. Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”. Certain alkyl groups include those containingbetween about one and about twenty five carbon atoms (e.g. methyl, ethyland the like).

The term “lower alkyl” generally refers to a straight, branched, orcyclic hydrocarbon chain containing 8 or fewer carbon atoms, and cancontain from 1 to 8, from 1 to 6, or from 1 to 4 carbon atoms. Certain“lower alkyl” groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and thelike. “Lower alkyls” can be optionally substituted at one or more carbonatoms of the hydrocarbon chain.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused to refer to those alkyl groups attached to the remainder of themolecule via an oxygen atom, an amino group, or a sulfur atom,respectively.

By “heteroatom” is meant atoms other than a carbon which may be presentin a carbon backbone or a linear, branched or cyclic compound. Certainheteroatoms include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P)and silicon (Si). Heteroatoms can be present in their reduced forms,e.g., —OH, —NH, and —SH.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, having the statednumber of carbon atoms and at least one heteroatom which can be a memberselected from O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally be quaternized. Normally heteroalkyl groups contain no morethan two heteroatoms linked in sequence. The heteroatom(s) 0, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Generally, up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ andCH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (usuallyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms which are members selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ where eachcan be independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ can beindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

The term “amino” or “amine group” refers to the group NR′R″ (or N⁺RR′R″)where R, R′ and R″ are independently selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted arylalkyl, heteroaryl, and substituted heteroaryl. A substituted amine is anamine group wherein R′ or R″ is other than hydrogen. In a primary aminogroup, both R′ and R″ are hydrogen, whereas in a secondary amino group,either, but not both, R′ or R″ is hydrogen. In addition, the terms“amine” and “amino” can include protonated and quaternized versions ofnitrogen, comprising the group —N⁺RR′R″ and its biologically compatibleanionic counterions.

The term “conjugate” refers to a molecule comprised of two or moremoieties bound together, optionally through a linking group, to form asingle structure. The binding can be made either by a direct connection(e.g., a chemical bond) between the components or by use of a linkinggroup. For example, a pregabalin conjugate generally refers to achemical compound composed of a pregabalin derivative covalently boundto a moiety of interest, which may be optionally linked through alinking group. In another example, a “pregabalin-enzyme conjugate”refers a pregabalin conjugate having an enzyme as the moiety ofinterest.

“Conjugation” refers to a process where two subunits are linked togetherto form a conjugate. The conjugation process can include one or moresteps, as described herein.

A “hapten” generally refers to a small molecule that can be specificallybound by an antibody but usually do not induce detectable or significantformation of antibodies unless bound to a carrier protein or other largeantigenic molecule. In the context of the present disclosure, pregabalinor a pregabalin derivative may be a hapten.

“Antigen”, as used herein, refers to a compound that binds specificallyto the variable region or binding site of an antibody. The term“antigen” and “immunogen” may in some cases be used interchangeably.

The term “epitope” refers to a region of an antigen that interacts withan antibody molecule. An antigenic molecule can have one or moreepitopes that can be recognized by the same or different antibodies. Anepitope or epitopic moiety may include a unique chemical configurationof an antigen, hapten or a reactive ligand. The chemical configurationmay be a linear sequence of chemical composition or even a spatial arrayof chemical groups in the chemical configuration. An epitope is thechemical configuration that associates directly with the binding site inthe antibody molecule. The antibody and the chemical group, hapten orreacting ligand containing the epitope form the “specific binding pair”(sbp).

A member of a specific binding pair (sbp member) is one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand), sbp member and sbp partner, or the like. In someembodiments, members of a specific binding pair are members of animmunological pair, such as antigen-antibody.

A “ligand” is an organic compound for which a receptor naturally existsor can be prepared. For example, the analyte may be a ligand andembodiments of the present disclosure provide methods for determiningthe concentration of the ligand.

A “receptor” is a compound or composition capable of recognizing aparticular spatial and polar organization of a molecule. These organizedareas of a molecule are referred to as epitopic or determinant sites.For example, receptors include, but are not limited to, antibodies andenzymes.

As used herein, the terms “immunogen” and “immunogenic” are meant torefer to substances capable of producing or generating an immuneresponse (e.g., antibody response) in an organism. An immunogen can alsobe antigen. In some embodiments, the immunogen has a high molecularweight (e.g., greater than 10,000). Thus, a variety of macromoleculessuch as proteins, lipoproteins, polysaccharides, nucleic acids andteichoic acids can be coupled to a hapten in order to form an immunogenin accordance with embodiments of the present disclosure.

As used herein, the term “immunogenicity” refers to the ability of amolecule to induce an immune response, which is determined both by theintrinsic chemical structure of the molecule and by whether or not thehost animal can recognize the compound. Small changes in the structureof an antigen can greatly alter the immunogenicity of a compound andhave been used extensively as a general procedure to increase thechances of raising an antibody, particularly against well-conservedantigens. For example, these modification techniques can alter regionsof the immunogen to provide better sites for T-cell binding or exposenew epitopes for B-cell binding.

A “carrier” or “immunogenic carrier” or “immunogenic moiety” as theterms are used herein, is an immunogenic substance, commonly apolypeptide, that can join with a hapten (such as a pregabalin moiety),thereby enabling the happen to induce an immune response and elicit theproduction of antibodies that can bind specifically with the antigen(hapten). Carrier substances include, but are not limited to, proteins,peptides (including polypeptides), glycoproteins, saccharides includingcomplex polysaccharides, particles, nucleic acids, polynucleotides, andthe like that are recognized as foreign and thereby elicit animmunologic response from the host.

The term “linker” or “linking group” as used in the present disclosurerefers to a chemical moiety that connects at least two substructures ofa compound, e.g., to provide for covalent connection between apregabalin hapten and a moiety of interest (e.g., a carrier ordetectable label). A linking group has at least one uninterrupted chainof atoms extending between the substructures. The atoms of a linkinggroup are themselves connected by chemical bonds. The number of atoms ina linking group is determined by counting the atoms other than hydrogen.Linking groups can include, but are not limited to, groups such asalkylene, heteroalkylene, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, amino, as described herein.

“Polypeptide” as used herein is meant to encompass a polyaminoacid ofany length, and encompasses proteins, protein fragments and peptides.Polypeptides may be genetically encoded or synthetically produced.Polypeptides may also be modified, e.g., by post-translational and/orchemical modification(s).

The term “antibody” includes a protein molecule having one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), andalpha (α) which encode the IgM, IgD, IgG, IgE, and IgA isotypes,respectively. “Antibody” as used herein is meant to include full lengthantibodies and antibody fragments, and may refer to a natural antibodyfrom any organism, an engineered antibody, or an antibody generatedrecombinantly for experimental, therapeutic, or other purposes asfurther defined below. Thus, the term “antibody raised against acompound” includes a synthesized antibody or compound having the samestructure as an antibody raised against the compound. The term“antibody” includes antibody fragments, as are known in the art, such asFab, Fab′, F(ab′)₂, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. The term“antibody” refers to both monoclonal and polyclonal antibodies.Antibodies can be antagonists, agonists, neutralizing, inhibitory, orstimulatory.

As used herein, the term “polyclonal antibody” refers to a heterogeneousmixture of antibodies with a wide range of specificities and affinitiesto a given antigen or epitope. Thus, the polyclonal antibody, which canalso be referred to as polyclonal antibodies, can include a plurality ofantibodies, each distinguishable from the others, that bind or otherwiseinteract with an antigen. The term “polyclonal” refers to antibodiesoriginating from multiple progenitor cells.

The different antibodies that comprise a polyclonal antibody can beproduced or generated by injecting an immunogen having an epitope intoan animal and, after an appropriate time, collecting and optionallypurifying the blood fraction containing the antibodies of interest. Inproducing antibodies, several parameters can be considered with respectto the final use for the polyclonal antibody. These parameters includethe following: (1) the specificity of the antibody (i.e., the ability todistinguish between antigens); (2) the avidity of the antibody (i.e.,the strength of binding an epitope); and (3) the titer of the antibody,which determines the optimal dilution of the antibody in the assaysystem.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies include hybrid and recombinant antibodiesproduced by splicing a variable (including hypervariable) domain of ananti-inhibitor antibody with a constant domain, or a light chain with aheavy chain, or a chain from one species with a chain from anotherspecies, or fusions with heterologous proteins, regardless of species oforigin or immunoglobulin class or subclass designation, as well asantibody fragments, e.g., Fab, F(ab)2, and Fv!, so long as they exhibitthe desired biological activity. For example, the monoclonal antibodiesmay be made by the hybridoma method first described by Kohler &Milstein, Nature, 256:495 (1975), or may be made by recombinant DNAmethods (U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may alsobe isolated from phage libraries generated using the techniquesdescribed in, e.g., McCafferty et al., Nature, 348:552-554 (1990).

The term “anti-pregabalin antibody” refers to antibodies that arecapable of specifically binding a pregabalin epitope of pregabalin, apregabalin derivative, or a pregabalin conjugate. “Anti-pregabalinantibodies” include both polyclonal and monoclonal antibodies, as wellas antigen-binding fragments thereof as defined above. A “pregabalinepitope” refers to an epitope that is present in pregabalin and in apregabalin derivative (e.g., a pregabalin conjugate).

The term “binds specifically” or “specifically binds” in the context ofantibody binding, refers to high avidity and/or high affinity binding ofan antibody to a specific antigen, e.g., to pregabalin. In specificbinding under appropriate conditions, antibody binding to pregabalin isstronger than binding of the same antibody to any other epitope,particularly those which may be present in molecules in associationwith, or in the same sample, as the pregabalin to be detected, e.g.,binds more strongly (e.g., higher affinity, higher avidity, or both) topregabalin than to a non-pregabalin epitope so that by adjusting bindingconditions the antibody binds almost exclusively to pregabalin, and notto non-pregabalin moieties that may be present in the sample. Antibodieswhich bind specifically to pregabalin may be capable of binding otherantigens at a weak, yet detectable, level (e.g., 10% or less of thebinding shown to pregabalin). Such weak binding, or background binding,is readily discernible from the specific antibody binding to pregabalin,e.g., by use of appropriate controls. “Antibody activity” or “antibodybinding activity” in the context of analyte binding assays generallyrefers to the ability of an antibody to bind a specific antigen inpreference to other potential antigens via the antigen combining sitelocated within a variable region of an immunoglobulin.

As used herein, a “detectable label” generally refers to an identifyingtag that can provide for a detectable signal, e.g., luminescence (e.g.,photoluminescence (e.g., fluorescence, phosphorescence),chemoluminescence (e.g., bioluminescence)), radioactivity,immunodetection, enzymatic activity, and the like).

By “detectably labeled antibody” an antibody (which, as defined above,includes antigen-binding fragments, etc.) having an attached detectablelabel. The detectable label may be attached by chemical conjugation, butwhere the label is a polypeptide, it could alternatively be attached bygenetic engineering techniques. Methods for production of detectablylabeled proteins are well known in the art. Detectable labels may beselected from a variety of such labels known in the art, but normallyare radioisotopes, fluorophores, enzymes (e.g., horseradish peroxidase),or other moieties or compounds which either emit a detectable signal(e.g., radioactivity, fluorescence, color) or emit a detectable signalafter exposure of the label to its substrate. Various detectablelabel/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine,avidin/streptavidin, luciferase/luciferin), methods for labellingantibodies, and methods for using labeled antibodies to detect anantigen are well known in the art.

“Antibody complex”, “antibody-antigen complex” generally refers to acomplex that results following specific binding of an antibody and itsantigen, e.g., between an anti-pregabalin antibody and pregabalin (or apregabalin derivative, e.g., pregabalin conjugate).

An “inhibitory antibody” is an antibody capable of inhibiting theactivity of an enzyme or an enzyme-ligand conjugate upon binding anepitope present on the enzyme. Inhibitory antibodies are distinguishedfrom anti-ligand antibodies capable of inhibiting the enzyme activity ofenzyme-ligand conjugates upon binding to the ligand.

The term “assessing” includes any form of measurement, and includesdetermining the presence or absence of an analyte. The terms“assessing”, “determining” (e.g., as in “determining the presence orabsence of”), “measuring”, “evaluating”, and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and/or determiningwhether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

Quantitative, semi-quantitative, and qualitative methods for determininganalyte are considered to be methods of measuring the amount of analyte.For example, a qualitative method which merely detects the presence orabsence of analyte in a sample suspected of containing an analyte isconsidered to be included within the scope of the embodiments. Synonymsfor the phrase “measuring the amount of analyte” which are contemplatedwithin the scope of the embodiments include, but are not limited to,detecting, measuring, or determining analyte; detecting, measuring, ordetermining the presence of analyte; and detecting, or determining theamount of analyte.

As used herein, the terms “immunoassay” or “immunodiagnostic” refer tolaboratory techniques or test systems that make use of the bindingbetween an antigen or analyte and an antibody in order to identifyand/or quantify at least one of the specific antigen or analyte orspecific antibody in a biological sample.

As used here, the term “competitive immunoassay” refers to anexperimental protocol in which a known amount of an identifiable antigenor analyte competes with another antigen or analyte for binding with anantibody. That is, a known antigen or analyte that binds with a knownantibody is combined with a sample that is suspected of containinganother antigen or analyte that also binds with the known antibody. Thisallows for the known antigen or analyte and another antigen or analyteto both compete for the binding site on the antibody. For example, apregabalin derivative that binds with an anti-pregabalin antibody can becombined with a sample suspected of containing pregabalin, and thepregabalin derivative and pregabalin compete for binding with theanti-pregabalin antibody. The competition for binding with the antibodycan then be used to determine whether or not pregabalin is present inthe sample, and can further be used to quantify the amount of pregabalinin the sample.

The term “accuracy” refers to the closeness of the agreement between theresult of a measurand and a true value of the measurand. The measurandis the substance measured or analyzed, such as the analyte or the ligandentering the binding reaction with the receptor or antibody.

The term “specificity” or “selectivity” refers to the preferentialbinding of a ligand to a receptor (e.g., antibody). Thus, specificitymay refer, in one embodiment, to the degree that pregabalin is boundselectively by an antibody. One measure of the specificity of a receptorto a ligand is crossreactivity. Compounds that cross-react are referredto as “crossreactants.” Crossreactants may include compounds havingsimilar chemical structures to pregabalin or a pregabalin derivative.Anti-pregabalin antibodies of the present disclosure include those thatbind an epitope of pregabalin, but that do not detectably bind acrossreactants of pregabalin.

The term “sensitivity” is used herein to describe a detection limit,e.g., the smallest amount of an analyte that results in a detectablesignal that is distinguishable from a signal obtained in the absence ofanalyte.

A “reagent” is a substance or compound that is added to a system inorder to bring about a chemical reaction, or added to see if a reactionoccurs.

As used herein, the term “isolated,” when used in the context of anisolated compound, antibody, conjugate, etc., refers to a compound ofinterest (e.g., a compound as described herein, a conjugate as describedherein, an antibody as described herein, etc.) that is in an environmentdifferent from that in which the compound naturally occurs. “Isolated”is meant to include compounds of interest (e.g., a compound as describedherein, a conjugate as described herein, or an antibody as describedherein) that are within samples that are substantially enriched for thecompound of interest and/or in which the compound of interest ispartially or substantially purified. As used herein, the term“substantially pure” refers to a compound of interest that is removedfrom its natural environment and is 60% or more free, 75% or more free,90% or more free, 95% or more free, 98% or more free, or 99% or morefree from other components with which it is naturally associated, and/orwith which it may be associated during synthesis or production.

DETAILED DESCRIPTION

Before embodiments of the present disclosure are described, it is to beunderstood that embodiments of the present disclosure are not limited toparticular embodiments described, and as such may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the embodiments. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within the embodiments, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the embodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the embodiments, some methods and materialsare now described. All publications mentioned herein are incorporatedherein by reference to disclose and describe the methods and/ormaterials in connection with which the publications are cited. It isunderstood that the present disclosure supersedes any disclosure of anincorporated publication to the extent there is a contradiction.

It is also noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aconjugate” includes a plurality of such conjugates and reference to “thesample” includes reference to one or more samples and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present applicationis not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

Pregabalin Derivatives

Pregabalin derivatives useful for pregabalin detection are provided inthe present disclosure. The term “pregabalin derivatives” is meant toencompass pregabalin-conjugates including immunogens and assay reagents(e.g., detectably labeled conjugates, such as enzyme conjugates,immobilized conjugates, and the like), as well as intermediates usefulin production of such pregabalin conjugates. In general, a pregabalinderivative is able to compete with pregabalin for binding to ananti-pregabalin antibody, e.g., in a pregabalin immunoassay. A schematicrepresentation of the structure of pregabalin is shown in FIG. 1 , panelA.

Pregabalin and gabapentin are anticonvulsants used in neurology,psychiatry and primary healthcare (FIG. 1 , panel A and FIG. 1 , panelB, respectively). Pregabalin and gabapentin inhibit calcium influx andsubsequent release of excitatory neurotransmitters; however, thecompounds differ in their pharmacokinetic and pharmacodynamiccharacteristics.

Pregabalin is the pharmacologically active S-enantiomer of3-aminomethyl-5-methylhexanoic acid. Embodiments of the presentdisclosure include a specific immunoassay for pregabalin that hassubstantially no crossreactivity to gabapentin. In certain embodiments,the chemical structural differences between pregabalin and gabapentinfacilitate a reduction in crossreactivity. For example, in certainembodiments, the 3-isobutyl group and/or the (S)-3-(aminomethyl) groupof pregabalin may be targeted by an anti-pregabalin antibody (FIG. 1 ,panel D). In certain embodiments, the 3-isobutyl group and/or the(S)-3-(aminomethyl) group of pregabalin are retained in haptens used toprepare immunogens and raise antibodies accordingly.

Structures of Pregabalin Derivatives

In certain embodiments, compounds (e.g., pregabalin derivatives) of thepresent disclosure have the general formula (A):

wherein

-   -   R¹ or R² is —X—W-L-Z, wherein        -   when R is —X—W-L-Z, then R² is —NH₂,        -   when R² is —X—W-L-Z, then R is —OH, and    -   X is NH;    -   W is selected from a bond, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,        substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl, and        carbonyl;    -   L is a bond or a linker; and    -   Z is selected from H, alkyl, substituted alkyl, a reactive        functional group, and a moiety of interest;    -   or a salt thereof.

As noted above, the term “linker” as used in the present disclosurerefers to a chemical moiety that connects at least two substructures ofa compound, e.g., to provide for covalent connection between apregabalin hapten and a moiety of interest (e.g., a carrier ordetectable label). In the context of a pregabalin conjugate, the linkercan be a chemical moiety that is the production of a reaction between areactive functional group and a moiety of interest, e.g., a polypeptide.Linkers include linear or branched, saturated or unsaturated,substituted or unsubstituted, hydrocarbon chains of from 1 to 40 carbonatoms, or from 1 to 20 carbon atoms, or from 1 to 15 carbon atoms, orfrom 1 to 10 carbon atoms, or from 1 to 6 carbon atoms, whichhydrocarbon chains may contain ring structures (e.g., up to two ringstructures) and 0-6 heteroatoms, or one or more heteroatoms.

In certain embodiments, W is a bond, such as a bond between X and L. Incertain embodiments, W is an alkyl or substituted alkyl. When W ofFormula (A) is an alkyl or substituted alkyl, the alkyl or substitutedalkyl may be a lower alkyl or substituted lower alkyl group. In someinstances, W can be, for example, a straight, branched, or cyclichydrocarbon chain containing 1 to 8 carbon atoms, from 1 to 6 carbonatoms, or from 1 to 4 carbon atoms.

In certain embodiments, W is selected from alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In certain embodiments, W isalkenyl or substituted alkenyl. In certain embodiments, W is alkynyl orsubstituted alkynyl. In certain embodiments, W is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, W is heterocyclyl orsubstituted heterocyclyl. In certain embodiments, W is aryl orsubstituted aryl. In certain embodiments, W is heteroaryl or substitutedheteroaryl.

In certain embodiments, when W of Formula (A) is a carbonyl group, thenL is a linker, which can be, for example, a hydrocarbon chain of from 1to 40 carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, or from 1 to 6 carbon atoms, which hydrocarbon chain mayoptionally contain ring structures (e.g., up to two ring structures) andone or more heteroatoms.

In certain embodiments, W is an alkyl (e.g., a lower alkyl) and L is alinker, such as, but not limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —C(O)(CH₂)_(n)—,    -   —C(O)(CH₂)_(n)NH—C(O)—,    -   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —C(O)—(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, W is a carbonyl, and L is a linker, such as, butnot limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n) NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, Z is H, alkyl, substituted alkyl, a reactivefunctional group, or a moiety of interest. In some embodiments, Z is H.In some embodiments, Z is alkyl or substituted alkyl, such as a C₁₋₁₀alkyl or C₁₋₁₀ substituted alkyl, or a C₁₋₆ alkyl or C₁₋₆ substitutedalkyl, or a C₁₋₃ alkyl or C₁₋₃ substituted alkyl.

In certain embodiments, Z is a reactive functional group. A reactivefunction group is a functional group capable of reacting with a reactivepartner to form a covalent bond. In some embodiments, when R¹ is—X—W-L-Z, then Z is a reactive functional group, and W is alkyl (e.g., alower alkyl). In some embodiments, when R² is —X—W-L-Z, then Z is areactive functional group, where the reactive functional group is not—OH or —COGH.

In some embodiments, when R¹ is —X—W-L-Z, then Z is a reactivefunctional group and —X—W-L-Z does not include a ring.

When Z of Formula (A) is a reactive functional group (e.g., a reactivefunctional group capable of reacting with a reactive partner to form acovalent bond), Z can be, for example a halogen (e.g., F, Br, Cl, I, andthe like), —OH, —SH, —NH₂, —O-lower alkyl, epoxy, —S-acyl, carboxyl,maleimidyl, haloacetamide, hydroxysuccinimidyl, succinimidyl, carbonate,anhydride, imidate, an isocyanate, an isothiocyanate, an imidoester, amaleimide, a thiolactone, a diazonium group, an acrylamide, an acylazide, an acyl nitrile, an alkyl halide, an aniline, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a halotriazine, ahydrazine, a hydrazide, an imido ester, a phosphoramidite, a reactiveplatinum complex, a sulfonyl halide, or a photoactivatable group.

In certain embodiments of Formula (A), Z is a moiety of interest. Forexample, moieties of interest for Z of Formula (A) include immunogeniccarriers (e.g., carrier proteins) and detectable labels. Moieties ofinterest are described in more detail below.

In some embodiments, the moiety of interest is a peptide, such as apolypeptide. In these embodiments, the moiety of interest is not asingle amino acid residue, but may be a polyamino acid chain (i.e.,polypeptide). In some embodiments, the moiety of interest is not a bileacid. In some embodiments where the moiety of interest is a polypeptide,the polypeptide is other than a transporter protein. In someembodiments, the pregabalin derivative is not a bile acid conjugate. Insome embodiments, the moiety of interest is not a bile acid.

In certain embodiments, the pregabalin derivative is a salt of apregabalin derivative. Salts of pregabalin derivatives include, but arenot limited to, alkali metal salts, such as (sodium salts, potassiumsalts, magnesium salts, etc.), halide salts (e.g., bromo, chloro, andthe like), acetate salts (e.g., salts with trifluoroacetic acid, and thelike), combinations thereof, and the like.

Pregabalin derivatives can be further described as having a formulaselected from Formula A1 and Formula A2:

wherein the wavy line (“

”) indicates the point at which the pregabalin moiety is attached to theremainder of the pregabalin derivative, e.g., attached through one ormore chemical moieties to a moiety of interest, e.g., a pregabalinconjugate, e.g., a pregabalin conjugate having a immunogenic carrier ora detectable label. For example, the wavy line can represent a site ofattachment to a polypeptide, detectable label, solid support, and thelike, where attachment can be through a chemical structure as set outabove.

Further examples of pregabalin derivatives are described below.

Pregabalin Derivatives of Formula (I)

In some embodiments, the pregabalin derivative is of Formula (I) below,in which the pregabalin derivative includes an extension from the aminegroup of pregabalin or its salt:

wherein W, L, and Z can be as defined above in Formula (A), and can beany combinations exemplified for Formula (A) as set out above.Accordingly, when W of Formula (I) is alkyl (e.g., a lower alkyl group),W can be, for example, a straight, branched, or cyclic hydrocarbon chaincontaining 1 to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4carbon atoms. In some instances, when W of Formula (I) is a carbonylgroup, then L is a linker, which can be, for example, a hydrocarbonchain of from 1 to 40 carbon atoms, or from 1 to 20 carbon atoms, orfrom 1 to 10 carbon atoms, or from 1 to 6 carbon atoms, whichhydrocarbon chain may optionally contain ring structures (e.g., up totwo ring structures) and one or more heteroatoms.

In certain embodiments, W is a bond, such as a bond between L and the—NH— group of the pregabalin derivative. In certain embodiments, W is analkyl or substituted alkyl. When W of Formula (I) is an alkyl orsubstituted alkyl, the alkyl or substituted alkyl may be a lower alkylor substituted lower alkyl group. In some instances, W can be, forexample, a straight, branched, or cyclic hydrocarbon chain containing 1to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms.

In certain embodiments, W is selected from alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In certain embodiments, W isalkenyl or substituted alkenyl. In certain embodiments, W is alkynyl orsubstituted alkynyl. In certain embodiments, W is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, W is heterocyclyl orsubstituted heterocyclyl. In certain embodiments, W is aryl orsubstituted aryl. In certain embodiments, W is heteroaryl or substitutedheteroaryl.

In certain embodiments, when W of Formula (I) is a carbonyl group, thenL is a linker, which can be, for example, a hydrocarbon chain of from 1to 40 carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, or from 1 to 6 carbon atoms, which hydrocarbon chain mayoptionally contain ring structures (e.g., up to two ring structures) andone or more heteroatoms.

In certain embodiments, W is an alkyl (e.g., a lower alkyl) and L is alinker, such as, but not limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —C(O)(CH₂)_(n)—,    -   —C(O)(CH₂)_(n)NH—C(O)—,    -   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —C(O)—(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, W is a carbonyl, and L is a linker, such as, butnot limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, Z is H, alkyl, substituted alkyl, a reactivefunctional group, or a moiety of interest. In some embodiments, Z is H.In some embodiments, Z is alkyl or substituted alkyl, such as a C₁₋₁₀alkyl or C₁₋₁₀ substituted alkyl, or a C₁₋₆ alkyl or C₁₋₆ substitutedalkyl, or a C₁₋₃ alkyl or C₁₋₃ substituted alkyl.

In certain embodiments, Z is a reactive functional group. When Z ofFormula (I) is a reactive functional group (e.g. a reactive functionalgroup capable of reacting with a reactive partner to form a covalentbond), Z can be, for example a halogen (e.g., F, Br, Cl, I, and thelike), —OH, —SH, —NH₂, —O-lower alkyl, epoxy, —S-acyl, carboxyl,maleimidyl, haloacetamide, hydroxysuccinimidyl, succinimidyl, carbonate,anhydride, imidate, an isocyanate, an isothiocyanate, an imidoester, amaleimide, a thiolactone, a diazonium group, an acrylamide, an acylazide, an acyl nitrile, an alkyl halide, an aniline, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a halotriazine, ahydrazine, a hydrazide, an imido ester, a phosphoramidite, a reactiveplatinum complex, a sulfonyl halide, or a photoactivatable group.Reactive functional groups for Z can be any of those as exemplified forZ of Formula (A) described herein.

In certain embodiments, Z is a moiety of interest. For example, moietiesof interest for Z of Formula (I) include immunogenic carriers (e.g.,carrier proteins) and detectable labels. Examples of moieties ofinterest are described in more detail as related to Formula (A) above.

In certain embodiments, —W-L-Z of Formula (I) may be selected from—(CH₂)_(m)—SH, —CO—(CH₂)_(n)—Br, and —(CH₂)_(o)—S—CH₂—CO—CH₂—Br, wherem, n, and o are each independently selected from 1, 2 or 3.

Examples of pregabalin derivatives of Formula (I) are provided in FIG. 2, such as pregabalin Hapten-A. In pregabalin Hapten-A, W is carbonyl, Lis —(CH₂)NH—C(O)(CH₂)—, and Z is Br. Salts of pregabalin derivative ofFormula (I) include those as described above for Formula (A).

Pregabalin Derivatives of Formula H

In some embodiments, pregabalin derivatives are characterized by anextension from the carbonyl group of pregabalin or its salt, which canbe described by Formula (II):

wherein W, L, and Z can be as defined above in Formula (A), and can beany combinations exemplified for Formula (A) as set out above.Accordingly, when W of Formula (II) is alkyl (e.g., a lower alkylgroup), W can be, for example, a straight, branched, or cyclichydrocarbon chain containing 1 to 8 carbon atoms, from 1 to 6 carbonatoms, or from 1 to 4 carbon atoms. In some instances, when W of Formula(II) is a carbonyl group, then L is a linker, which can be, for example,a hydrocarbon chain of from 1 to 40 carbon atoms, or from 1 to 20 carbonatoms, or from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms, whichhydrocarbon chain may optionally contain ring structures (e.g., up totwo ring structures) and one or more heteroatoms.

In certain embodiments, W is a bond, such as a bond between L and the—NH— group of the pregabalin derivative. In certain embodiments, W is analkyl or substituted alkyl. When W of Formula (II) is an alkyl orsubstituted alkyl, the alkyl or substituted alkyl may be a lower alkylor substituted lower alkyl group. In some instances, W can be, forexample, a straight, branched, or cyclic hydrocarbon chain containing 1to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms.

In certain embodiments, W is selected from alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In certain embodiments, W isalkenyl or substituted alkenyl. In certain embodiments, W is alkynyl orsubstituted alkynyl. In certain embodiments, W is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, W is heterocyclyl orsubstituted heterocyclyl. In certain embodiments, W is aryl orsubstituted aryl. In certain embodiments, W is heteroaryl or substitutedheteroaryl.

In certain embodiments, when W of Formula (II) is a carbonyl group, thenL is a linker, which can be, for example, a hydrocarbon chain of from 1to 40 carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, or from 1 to 6 carbon atoms, which hydrocarbon chain mayoptionally contain ring structures (e.g., up to two ring structures) andone or more heteroatoms.

In certain embodiments, W is an alkyl (e.g., a lower alkyl) and L is alinker, such as, but not limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —C(O)(CH₂)_(n)—,    -   —C(O)(CH₂)_(n)NH—C(O)—,    -   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —C(O)—(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, W is a carbonyl, and L is a linker, such as, butnot limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, Z is H, alkyl, substituted alkyl, a reactivefunctional group, or a moiety of interest. In some embodiments, Z is H.In some embodiments, Z is alkyl or substituted alkyl, such as a C₁₋₁₀alkyl or C₁₋₁₀ substituted alkyl, or a C₁₋₆ alkyl or C₁₋₆ substitutedalkyl, or a C₁₋₃ alkyl or C₁₋₃ substituted alkyl.

In certain embodiments, Z is a reactive functional group. When Z ofFormula (II) is a reactive functional group (e.g., a reactive functionalgroup capable of reacting with a reactive partner to form a covalentbond), Z can be, for example a halogen (e.g., F, Br, Cl, I, and thelike), —OH, —SH, —NH₂, —O-lower alkyl, epoxy, —S-acyl, carboxyl,maleimidyl, haloacetamide, hydroxysuccinimidyl, succinimidyl, carbonate,anhydride, imidate, an isocyanate, an isothiocyanate, an imidoester, amaleimide, a thiolactone, a diazonium group, an acrylamide, an acylazide, an acyl nitrile, an alkyl halide, an aniline, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a halotriazine, ahydrazine, a hydrazide, an imido ester, a phosphoramidite, a reactiveplatinum complex, a sulfonyl halide, or a photoactivatable group.Reactive functional groups for Z can be any of those as exemplified forZ of Formula (A) described herein.

In certain embodiments, Z is a moiety of interest. For example, moietiesof interest for Z of Formula (II) include immunogenic carriers (e.g.,carrier proteins) and detectable labels. Examples of moieties ofinterest are described in more detail below as related to Formula (A)above.

In certain embodiments, —W-L-Z of Formula (II) may be selected from—(CH₂)_(m)—SH, —CO—(CH₂)_(n)—Br, or —(CH₂)_(o)—S—CH₂—CO—CH₂—Br, where m,n, and o are each independently selected from 1, 2 or 3.

Examples of pregabalin derivatives of Formula (II) are provided in FIG.2 , such as pregabalin Hapten-B. In pregabalin Hapten-B, W is a bond, Lis —(CH₂)₂NH—C(O)(CH₂)—, and Z is Br.

Salts of pregabalin derivatives of Formula (II) include those asdescribed above for Formula (A).

Methods of Making Pregabalin Derivatives

The compounds of the present disclosure are synthesized by anappropriate combination of generally known synthetic methods. Techniquesuseful in synthesizing the compounds of the present disclosure aregenerally known those of skill in the relevant art. The discussion belowis offered to illustrate examples of certain methods available for usein synthesizing the compounds of the present disclosure, and is notintended to limit the scope of reactions or reaction sequences that areuseful in preparing the compounds of the present disclosure.

In certain embodiments, compounds of the present disclosure aresynthesized as described herein. For example, Hapten-A, which includesan amine modified derivative of pregabalin may be synthesized accordingto Scheme 1 as described below.

In certain embodiments, pregabalin may be taken into an aqueous solutionof sodium carbonate and cooled in ice bath. A solution of Fmoc-Cl indioxane may be added. The reaction can be allowed to warm up to roomtemperature and stirred. In certain embodiments, water is added andextracted with ethyl acetate. The water layer may be cooled (e.g., in anice bath) and acidified to pH=1 with an acid (e.g., HCl), then extractedwith ethyl acetate (3×50 ml). The combined organic layer may be washedwith brine, dried over MgSO₄, and concentrated to give a crude product.The crude product may be purified on silica gel using ethyl acetate inhexane to give intermediate compound 2.

In certain embodiments, Fmoc-pregabalin (compound 2) andN,N-diisopropylethylamine (DIEA) are mixed in dichloromethane (DCM) andadded to 2-chlorotrityl resin. After one hour of shaking, methanol maybe added and mixed. Resulting compound 3 may be drained, washedsuccessively with DCM and dimethylformamide (DMF). In certainembodiments, a solution of piperidine in DMF is added to compound 3 andshaken. The reactions may be drained and washed as described above. Incertain embodiments, compound 3 is dried under vacuum and used for thenext step.

In certain embodiments, Fmoc-glycine and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) are added to a mixture of compound 3in DMF. In certain embodiments, DIEA is added and shaken. The reactionmay be repeated twice. In certain embodiments, the resin is drained andwashed with DMF, DCM and MeOH. A solution of piperidine/DMF may be addedand shaken. In certain embodiments, resulting compound 4 is drained andwashed as described above and dried under vacuum.

In certain embodiments, to a solution of 2-bromoacetic acid in dry DMFis added N,N′-diisopropylcarbodiimide (DIC). In certain embodiments,compound 4 is added to the mixture and shaken. This reaction may berepeated twice. The resulting crude product may be drained and washedwith DMF, DCM, and methanol. The crude product may then be dried undervacuum. In certain embodiments, the crude product is taken in DCM andtrifluoroacetic acid (TFA) is added and shaken. The crude product may bedrained and washed with TFA/DCM solution. The combined drained and washsolution may be evaporated to dryness and triturated with ether. Incertain embodiments, the crude product is purified on silica gel usingmethanol in DCM to give an amine modified derivative of pregabalin,Hapten-A.

In certain embodiments, Hapten-B, which includes a carboxylic acidmodified derivative of pregabalin may be synthesized according to Scheme2 as described below.

In certain embodiments, pregabalin is dissolved in a mixture of waterand dioxane, then 1N NaOH is added and cooled to 5° C. Bocanhydride maythen be added and stirred in an ice bath. The reaction mixture may beallowed to warm up to room temperature and stirred. In certainembodiments, dioxane is evaporated under vacuum. Water may be added,cooled, and acidified to pH=2-3. The mixture may be extracted with ethylacetate. In certain embodiments, the organic layer is washed with water,dried over MgSO₄ and concentrated to give intermediate compound 5.

Intermediate compound 5 may be dissolved in DCM. DIC andhydroxybenzotriazole (HOBt) may be added and allowed to react for 5 minat room temperature. In certain embodiments, alloc-1,2-diaminoethane isadded. In certain embodiments, after stirring, solvent is evaporated,water is added and extracted with ethyl acetate, washed with brine anddried over MgSO₄. After evaporation of solvent, crude product may beobtained, which can be purified on silica gel using methanol in DCM togive compound 6.

In certain embodiments, compound 6 is dissolved in DCM.Dichlorobis(triphenylphosphine) palladium(II) can be added followed bytributyltinhydride and acetic acid. After overnight stirring, morepalladium catalyst may be added and stirred. In certain embodiments, thepresence or absence of starting material is detected by thin layerchromatography (TLC). Solvent may be evaporated and the crude productcan be triturated in ether and decanted. In certain embodiments, thecrude product is left under high vacuum for 2 hrs. This material canthen be dissolved in DCM and hexane and left at 4° C. overnight. Incertain embodiments, solvent is decanted and the solid colorlessprecipitate left under vacuum to give compound 7. In certainembodiments, compound 7 may be used in the next step without furtherpurification.

In certain embodiments, compound 7 in DCM is cooled and triethylamine isadded, followed by bromoacethyl bromide. The reaction mixture may bestirred in an ice bath until no starting material remains (e.g., asmonitored by TLC). In certain embodiments, solvent is removed undervacuum, and water is added and extracted with ethyl acetate. In certainembodiments, the organic layer is dried over MgSO₄ and concentrated togive crude product, which can be purified on silica gel using ethylacetate/hexanes to give compound 8.

In certain embodiments, to a solution of compound 8 in DCM, is added TFAat room temperature and stirred. In certain embodiments, after TLC showsno remaining starting material, solvent is evaporated under vacuum andthe crude product is triturated with ether. In certain embodiments, theproduct is left under vacuum to give a carboxylic acid modifiedderivative of pregabalin, Hapten-B.

Pregabalin Conjugates

In certain embodiments, a pregabalin conjugate includes a pregabalinmoiety and a covalently bound moiety of interest, where the pregabalinmoiety and moiety of interest can be covalently bound as a result ofreaction through a reactive functional group of the pregabalinderivative. Pregabalin conjugates of the present disclosure are thus ofthe general Formula (A′):

wherein

-   -   R¹ or R² is —X—W-L-Z, wherein        -   when R¹ is —X—W-L-Z, then R² is —NH₂,        -   when R² is —X—W-L-Z, then R¹ is —OH, and    -   X is NH;    -   W is selected from a bond, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,        substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl, and        carbonyl;    -   L is a bond or a linker; and    -   Z is a moiety of interest (e.g., an immunogenic carrier or a        detectable label);    -   or a salt thereof.

In some instances, when W of Formula (A′) is alkyl (e.g., a lower alkylgroup), W can be, for example, a straight, branched, or cyclichydrocarbon chain containing 1 to 8 carbon atoms, from 1 to 6 carbonatoms, or from 1 to 4 carbon atoms. In some instances, when W of Formula(A′) is a carbonyl group, then L is a linker, which can be, for example,a hydrocarbon chain of from 1 to 40 carbon atoms, or from 1 to 20 carbonatoms, or from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms, whichhydrocarbon chains may contain ring structures (e.g., up to two ringstructures) and one or more heteroatoms.

In certain embodiments, W is selected from alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In certain embodiments, W isalkenyl or substituted alkenyl. In certain embodiments, W is alkynyl orsubstituted alkynyl. In certain embodiments, W is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, W is heterocyclyl orsubstituted heterocyclyl. In certain embodiments, W is aryl orsubstituted aryl. In certain embodiments, W is heteroaryl or substitutedheteroaryl.

In certain embodiments, when W of Formula (A′) is a carbonyl group, thenL is a linker, which can be, for example, a hydrocarbon chain of from 1to 40 carbon atoms, or from 1 to 20 carbon atoms, or from 1 to 10 carbonatoms, or from 1 to 6 carbon atoms, which hydrocarbon chain mayoptionally contain ring structures (e.g., up to two ring structures) andone or more heteroatoms.

In certain embodiments, W is an alkyl (e.g., a lower alkyl) and L is alinker, such as, but not limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —C(O)(CH₂)_(n)—,    -   —C(O)(CH₂)_(n)NH—C(O)—,    -   —C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—,    -   —C(O)—(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments, W is a carbonyl, and L is a linker, such as, butnot limited to, the following:

-   -   —(CH₂)_(n)C(O)—,    -   —(CH₂)_(n)SCH₂C(O)—,    -   —(CH₂)_(m)SCH₂—C(O)(CH₂)_(n)—,    -   —(CH₂)_(m)C(O)NH(CH₂)_(n)—,    -   —(CH₂)_(n)NH—C(O)—,    -   —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, and    -   —(CH₂)_(n)—;        wherein m and n are each independently selected from an integer        from 0 to 10, such as an integer from 1 to 10, or from 1 to 6,        or from 1 to 3.

In certain embodiments of Formula (A′), Z is a moiety of interest. Forexample, moieties of interest for Z of Formula (A′) include immunogeniccarriers (e.g., carrier proteins) and detectable labels. Moieties ofinterest are described in more detail below.

In some embodiments, the moiety of interest is a peptide, such as apolypeptide. In these embodiments, the moiety of interest is not asingle amino acid residue, but may be a polyamino acid chain (i.e.,polypeptide). In some embodiments, the moiety of interest is not a bileacid. In some embodiments where the moiety of interest is a polypeptide,the polypeptide is other than a transporter protein. In someembodiments, the pregabalin derivative is not a bile acid conjugate. Insome embodiments, the moiety of interest is not a bile acid.

In certain embodiments, the pregabalin derivative is a salt of apregabalin derivative. Salts of pregabalin derivatives include, but arenot limited to, alkali metal salts, such as (sodium salts, potassiumsalts, magnesium salts, etc.), halide salts (e.g., bromo, chloro, andthe like), acetate salts (e.g., salts with trifluoroacetic acid, and thelike), combinations thereof, and the like.

In certain embodiments, the moiety of interest is bound to thepregabalin conjugate through a linking group selected from —CONH—,—NHCO—, —NHCONH—, —NH—(C═S)—NH—, —O—CO—NH—, —NH—O—CO—, —S—, —NH—(C═NH)—,—N═N—, and —NH—.

The present disclosure also provides pregabalin conjugates of theFormula (I′):

wherein W and L are as defined in Formula (A′) above, and Z is a moietyof interest.

In some instances, linkers for pregabalin conjugates of Formula (I′) areas described above in Formula (A′). Salts of pregabalin conjugate ofFormula (I′) can include those as described for Formula (A′) above.

Examples of pregabalin conjugates of Formula (I′) are provided in FIG. 5and FIG. 7 , such as pregabalin conjugates KLH-A and rG6PDH-A. InConjugates KLH-A and rG6PDH-A, W is a carbonyl, L is—(CH₂)(NH)(C═O)(CH₂)—, and Z is a carrier protein (e.g., KLH), which isattached to the pregabalin moiety through a sulfhydryl group of theprotein or a sulfhydryl of a cysteine residue of the protein (e.g.,through a sulfide bond).

In certain embodiments, pregabalin conjugates are characterized by anextension from the carbonyl group of pregabalin, which can be describedby Formula (II′):

wherein W and L are as defined in Formula (A′) above, and Z is a moietyof interest.

In some instances, linkers for pregabalin conjugates of Formula (II′)are as described above in Formula (A′). Salts of pregabalin conjugatesof Formula (II′) can include those as described above for Formula (A′).

Examples of pregabalin conjugates of Formula (II′) are provided in FIG.6 , FIG. 8 and FIG. 9 , such as Conjugates KLH-B, G6PDH-B2, andrG6PDH-B1, respectively. In Conjugates KLH-B, rG6PDH-B1 and G6PDH-B2, Wis a bond, L is —(CH₂)₂(NH)—(C═O)(CH₂)—, and Z is a carrier protein(e.g., KLH), which is attached to the pregabalin moiety through asulfhydryl group of the protein or a sulfhydryl of a cysteine residue ofthe protein (e.g., through a sulfide bond).

In certain embodiments, where the moiety of interest has multipleavailable covalent attachment sites for a pregabalin moiety (e.g., areactive partner having multiple reaction sites for reaction with apregabalin derivative), the pregabalin conjugate can include more thanone pregabalin moiety. Accordingly, pregabalin conjugates of the presentdisclosure include those in which two or more, pregabalin moieties arebound to the same moiety of interest (e.g., polypeptide (e.g., carrierprotein), solid support (e.g., Sepharose® bead, particle (e.g., goldparticle, magnetic particle)). Such pregabalin conjugates can berepresented by Formula (I′″) or Formula (II′″) as follows:

wherein W and L are as defined in Formula (A′) above, and Z is a moietyof interest having two or more attachment sites for a pregabalin moiety;or

wherein W and L are as defined in Formula (A′) above, and Z is a moietyof interest having two or more attachment sites for a pregabalin moiety;andwherein q is 1 or more, such as 2 or more, 5 or more, or 10 or more, 15or more, up to a number of reaction sites available on Z. For example,where Z is a polypeptide, q can be up to the number of accessible aminoacid residues reactive with a pregabalin derivative having anappropriate reactive functional group.

Moieties of Interest of Pregabalin Conjugates

In certain embodiments, the moiety of interest of pregabalin conjugatesof the present disclosure can be any suitable chemical entity orsupport, such as one adapted for use in an assay, or for generatingreagents useful in such assays (e.g., anti-pregabalin antibodyproduction), described herein.

Accordingly, in some instances, the moiety of interest can be, forexample, an immunogenic carrier, a detectable label, or a support.

Examples of immunogenic carriers include, but are not limited to,polypeptides (which term is used to encompass amino acid chains of anylength, including peptides and proteins), modified polypeptides (e.g.,post-translationally and/or chemically modified, e.g., lipoproteins,glycoproteins, and the like), and polysaccharides. In certainembodiments, the immunogenic carrier is a polypeptide immunogeniccarrier.

Further examples of moieties of interest include detectable labels, suchas, but not limited to, polypeptides having an immunodetectable epitope(e.g., detectable by binding of a binding partner that specificallybinds the epitope (e.g., an HA tag)), nucleic acids (which can bedetectable by use of a hybridization probe or by PCR-based methods),radioactive isotopes, enzymes (including enzyme fragments, enzyme donorfragments, enzyme acceptor fragments, coenzymes), enzyme ligands (e.g.,enzyme substrates, enzyme inhibitors), fluorescent moieties (includingfluorophores and quenchers), phosphorescent moieties, anti-stokesup-regulating moieties, chemiluminescent moieties, luminescent moieties,chromophores, radioactive isotopes, and combinations thereof.

Further examples of moieties of interest include a support, such as, butnot limited to, solid supports (e.g., arrays), particles (including goldparticles, microparticles, magnetic particles, beads, and the like), andliposomes, and combinations thereof.

As described above, a “detectable label” generally refers to anidentifying tag that can provide for a detectable signal, e.g.,luminescence (e.g., photoluminescence (e.g., fluorescence,phosphorescence), chemoluminescence (e.g., bioluminescence)),radioactivity, immunodetection, enzymatic activity, and the like).Examples of a label include a polypeptide such as an antigen, enzyme, anantibody, a nucleic acid, a fluorophor, a quencher (e.g., of a FRETpair), a phosphorescent group, a chemiluminescent group, a chromophoricgroup, an electrochemically active group, an electrochemiluminescentgroup, a group that undergoes a change in fluorescence, phosphorescence,chemiluminescence or electrochemical property upon binding (e.g., asdescribed in U.S. Pat. Nos. 6,203,974 and 6,159,750), a solid particle,a gold particle, a radioactive isotope, an enzyme ligand (e.g., anenzyme inhibitor, an enzyme substrate), an enzyme cofactor, a member ofan enzyme donor-acceptor pair, and the like.

The detectable label can be a non-isotopic signal-generating moiety.”“Non-isotopic signal-generating moiety”, as used herein, refers to amoiety that does not emit radioactivity as a detectable signal. By wayof example, a non-isotopic signal-generating moiety is an enzyme,fluorescent compound, or a luminescent compound.

Other examples of moieties of interest are further described below.

Pregabalin-Carrier Protein Conjugates

In some embodiments, the moiety of interest is an immunogenic carrier,such as an immunogenic carrier protein. Such pregabalin conjugates finduse in production of anti-pregabalin antibodies, which in turn find usein the pregabalin detection assays described herein. Examples ofcarriers include, but are not limited to, proteins, glycoproteins,complex polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host.

Various protein types may be employed as a poly(amino acid) immunogeniccarrier. These types include albumins, serum proteins, e.g., globulins,ocular lens proteins, lipoproteins, etc. Examples of proteins includebovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), eggovalbumin, bovine gamma-globulin (BGG), etc. Immunogenic polypeptidesinclude genetically-encodable and synthetic polypeptides, and the like.

The immunogenic carrier can also be a polysaccharide, which includeshigh molecular weight polymers built up by repeated condensations ofmonosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide can also contain polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides. The immunogenic carrier can also be a particle. Incertain embodiments, the particles are 0.01 microns or more in diameter,such as 0.01 micron to 100 micron, for example 0.05 micron to 10 micronin diameter. The particle can be organic or inorganic, swellable ornon-swellable, porous or non-porous, optionally of a densityapproximating water, such as from about 0.7 to 1.5 g/mL, and composed ofmaterial that can be transparent, partially transparent, or opaque. Theparticles can be biological materials such as cells and microorganisms,including non-limiting examples such as erythrocytes, leukocytes,lymphocytes, hybridomas, Streptococcus, Staphylococcus aureus, E. coli,and viruses. The particles can also include organic and inorganicpolymers, liposomes, latex, phospholipid vesicles, or lipoproteins, orcombinations thereof.

Immunogenic carriers described herein include keyhole limpet hemocyanin(KLH) and bovine serum albumin (BSA). FIG. 5 and FIG. 6 show schematicrepresentation of examples of pregabalin conjugates having a pregabalinmoiety linked to KLH. Dimerized pregabalin conjugates can be linkedthrough a disulfide bond, which may be reduced to generate monomericpregabalin conjugates, e.g., by reaction with dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP). Pregabalin carrier proteinconjugates may include a plurality of pregabalin derivatives covalentlybonded to the protein carrier, as discussed in Formula (I′), and Formula(II′″), above.

Pregabalin-Enzyme Conjugates

In certain embodiments, the moiety of interest of the pregabalinconjugate is an enzyme. In some instances, the enzyme can serve as anon-isotopic signal generating moiety, and may be any enzyme thatprovides for a detectable signal useful in, for example, an immunoassaydescribed herein. Examples of enzymes include, but are not limited to,alkaline phosphatase, β-galactosidase, horse radish peroxidase,glucose-6-phosphate dehydrogenase (G6PDH), and the like. The G6PDHrefers to both a naturally occurring G6PDH and G6PDH variants thatcontain one or more cysteine residues non-native to naturally-occurringG6PDH (e.g., recombinant G6PDH, rG6PDH).

In some embodiments, pregabalin enzyme conjugates provide an immunoassayreagent that competes with pregabalin that may be present in a samplefor binding to an anti-pregabalin antibody, where the presence orabsence of a detectable signal provided by the enzyme is indicative ofthe presence or absence of a pregabalin-anti-pregabalin antibodycomplex. Pregabalin enzyme conjugates containing G6PDH (includingnaturally occurring G6PDH and G6PDH variants, such as G6PDH cysteinevariants) find use in such assays.

FIG. 7 , FIG. 8 and FIG. 9 show schematic representations of examples ofpregabalin-enzyme conjugates, such as pregabalin rG6PDH, and G6PDHconjugates. Dimerized pregabalin-enzyme conjugates can be linked througha disulfide bond, which may be reduced to generate monomeric pregabalinenzyme conjugates, e.g., by reaction with DTT or TCEP. Pregabalin enzymeconjugates may include a plurality of pregabalin derivatives covalentlybonded to the protein carrier, as discussed in Formulae (I′″), and(II′″) above.

Other Pregabalin Conjugates

The moiety of interest may be a support, thus immobilizing thepregabalin conjugate. The moiety of interest may provide a detectablesignal, such as a fluorophore, a fluorescence quencher, a radioisotope,and metal particle (e.g., in SERS-based assays). Pregabalin conjugatescan include, for example, a first member of a FRET pair (e.g., a memberof a fluorophore/quencher pair), where the pregabalin conjugate is usedin connection with an anti-pregabalin antibody having the second memberof the FRET pair, such that binding of the labeled anti-pregabalinantibody to the labeled pregabalin conjugate in a complex provides for adetectable signal different from that when the labeled anti-pregabalinantibody and labeled pregabalin conjugate are not in a complex with oneanother (e.g., as when binding of pregabalin blocks binding of theantibody to the pregabalin conjugate).

Methods of Making Pregabalin Conjugates

In certain embodiments, pregabalin conjugates are prepared bysynthesizing a pregabalin derivative having a reactive functional group(e.g., as described above), and reacting the pregabalin derivative witha reactive partner (e.g., a protein) under conditions that permit aconjugation reaction to occur, and then isolating the conjugate from thereaction mixture.

For example, a protein conjugate can be prepared by combining an excessof a bromoacetyl adduct with a protein having one or more free thiolgroups as described in U.S. Pat. No. 6,455,288, the disclosure of whichis incorporated herein by reference. Free sulfhydryl groups may beprovided in the form of free cysteine residues or by reducing proteindisulfide bonds using a reducing reagent, such as dithiothreitol (DTT).Alternatively, thiol groups can be added to a protein having freeprimary amino groups by reacting with 2-iminothiolane (IT) in aqueousbuffer, followed by removal of unreacted IT. Examples of protocols forthe thiolation of the protein KLH are provided in U.S. Pat. No.5,439,798, the disclosure of which is incorporated herein by reference.Other examples of methods of protein conjugation methods are furtherdescribed in U.S. Pat. Nos. 6,033,890, 6,090,567, and 6,455,288, thedisclosures of each of which are incorporated herein by reference.

FIG. 3 and FIG. 4 show examples of thiolation of KLH and native G6PDH,respectively. FIG. 5 , FIG. 6 , FIG. 7 , FIG. 8 and FIG. 9 are schematicrepresentations of synthesis schemes of examples of pregabalin-enzymeconjugates (e.g., rG6PDH and native G6PDH conjugates), andpregabalin-immunogenic carrier conjugates using KLH.

Anti-Pregabalin Antibodies

As noted above, the term “antibody” as used in the context of thepresent disclosure, refers to a specific binding partner of an analyte(e.g., pregabalin), and is meant to encompass whole antibodies as wellas antigen-binding fragments thereof (such as, for example, F(ab′)₂,Fab′, Fab and Fv), naturally occurring antibodies, hybrid antibodies,chimeric antibodies, single-chain antibodies, and antibody fragmentsthat retain antigen binding specificity, and the like. Antibodies can beof any class (e.g., IgM, IgG, IgA, IgE; frequently IgG) and generatedfrom any source (although usually non-human, usually a non-human mammalsuch as a rabbit, mouse, rat, goat, etc.). Thus, “antibody” is meant toencompass not only intact immunoglobulin molecules, but also suchfragments and derivatives of immunoglobulin molecules as may be preparedby techniques known in the art, and retaining the antibody activity ofan intact immunoglobulin.

Antibodies may be derived from polyclonal compositions or monoclonalcompositions. As noted above, “antibodies” is also meant to encompasssingle chain antibodies or scFvs, where such recombinantly producedantibody fragments retain the binding characteristics of the aboveantibodies. Recombinantly produced antibody fragments within the meaningof “antibody” generally include at least the VH and VL domains of thesubject antibodies, so as to retain the binding characteristics of thesubject antibodies. These recombinantly produced antibody fragments maybe readily prepared using any convenient methodology, such as themethodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371, thedisclosures of which are herein incorporated by reference.

Anti-pregabalin antibodies include those that bind one or morepregabalin epitopes. Anti-pregabalin antibodies thus include antibodiesthat bind, particularly that specifically bind, one or more of an amineepitope of a pregabalin moiety, a carboxylic acid epitope of apregabalin moiety, or any combination thereof (e.g., both an amineepitope and a carboxylic acid epitope). Anti-pregabalin antibodies maybind one or more of unconjugated pregabalin, a pregabalin derivative, apregabalin conjugate, or any combination thereof.

Producing Anti-Pregabalin Antibodies

Anti-pregabalin antibodies can be prepared by using an immunogenicpregabalin conjugate described herein and applying methods for antibodyproduction that are known in the art. For examples of general techniquesused in raising, purifying and modifying antibodies, and the design andexecution of immunoassays, the reader is referred to Handbook ofExperimental Immunology (D. M. Weir & C. C. Blackwell, eds.); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); David Wild,ed., The Immunoassay Handbook (Stockton Press N.Y., 1994); and R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Methods ofImmunological Analysis (Weinheim: VCH Verlags gesellschaft mbH, 1993).

Antibodies obtained using any of the disclosed techniques are screenedor purified not only for their ability to react with pregabalin, but fora low cross-reactivity with potential interfering substances.“Cross-reactivity” may be determined in a quantitative immunoassay byestablishing a standard curve using known dilutions of the targetanalyte, pregabalin. The standard curve is then used to calculate theapparent concentration of the interfering substance present in variousknown amounts in samples assayed under similar condition. Thecross-reactivity is the apparent concentration divided by the actualconcentration multiplied by 100. An exemplary immunoassay fordetermining cross-reactivity is a homogeneous enzyme immunoassay using awild type G6PDH as described in U.S. Pat. No. 3,817,837 or mutant G6PDHengineered to contain a cysteine per subunit as described in U.S. Pat.Nos. 6,033,890, 6,090,567 and 6,455,288. Furthermore, thecross-reactivity can be determined in the same type of immunoassay inwhich the antibody will ultimately be used.

In certain embodiments, the subject immunoassays (e.g., the subjectanti-pregabalin antibodies) have a crossreactivity for interferingsubstances (e.g., crossreactants), such as gabapentin, or amino acids(e.g., leucine), that is 10% or less, or 5% or less, or 1% or less, or0.5% or less, or 0.1% or less, or 0.05% or less, or 0.01% or less, or0.005% or less, or 0.001% or less.

Producing Polyclonal Antibodies

Polyclonal antibodies that bind pregabalin may be raised byadministration of an immunogenic pregabalin conjugate to an animal host,usually mixed with an adjuvant. Any animal host which producesantibodies can be used. The immunogen may be prepared for injection byrehydrating lyophilized immunogen to form a solution or suspension.Examples of adjuvants include water-in-oil immersions, such as Freund'scomplete adjuvant for the first administration, and Freund's incompleteadjuvant for booster doses. The preparation is typically administered ina variety of sites, and typically in two or more doses over a course ofat least 4 weeks. Serum is harvested and tested for the presence ofanti-pregabalin antibody using a pregabalin-protein conjugate or otherpregabalin conjugates in a standard immunoassay or precipitationreaction.

Polyclonal antisera typically contain antibodies not reactive withpregabalin and cross-reactive with other substances. Methods forpurifying specific antibodies from a polyclonal antiserum are known inthe art. An example of a method is affinity purification using a columnof pregabalin conjugated to a solid phase. One manner of preparing apregabalin column is to conjugate pregabalin or a pregabalin derivativeto a protein other than the protein used in the immunogen, and thenattach the conjugate to a commercially available activated resin, suchas CNBr-activated SEPHAROSE™. The anti-pregabalin antibody is passedover the column, the column is washed, and the antibody is eluted with amild denaturing buffer such as 0.1 M glycine, 0.2 M NaCl, pH 2.5.

Producing Monoclonal Antibodies

Anti-pregabalin monoclonal antibodies may be prepared by a number ofdifferent techniques. For example, for hybridoma technology, techniquesfor producing monoclonal antibodies are described in Harrow E, Lane D.,1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, U.S. Pat. Nos. 4,491,632, 4,472,500, and4,444,887, and Methods in Enzymology, 73B:3 (1981). In certainembodiments, monoclonal antibodies are produced by immortalizing andcloning a splenocyte or other antibody-producing cell recovered from ananimal that has been immunized against pregabalin as described earlier.The clone may be immortalized by a procedure such as fusion with anon-producing myeloma, by transfecting with Epstein Barr Virus, ortransforming with oncogenic DNA. The treated cells may be cloned andcultured, and clones may be selected that produce antibody of thedesired specificity. Specificity testing may be performed on culturesupernatants by a number of techniques, such as using the immunizingantigen as the detecting reagent in an immunoassay. A supply ofmonoclonal antibody from the selected clone can then be purified from alarge volume of culture supernatant, or from the ascites fluid ofsuitably prepared host animals injected with the clone. The antibody maybe tested for activity as raw supernatant or ascites, and is optionallypurified using biochemical preparation techniques, such as, but notlimited to, ammonium sulfate precipitation, ion exchange chromatography,and gel filtration chromatography.

Producing Fragments and Other Derivatives of Immunoglobulins

Fragments and other derivatives of immunoglobulins can be prepared bymethods of standard protein chemistry, for example, subjecting theantibody to cleavage with a proteolytic enzyme such as pepsin, papain,or trypsin; and reducing disulfide bonds with such reagents asdithiothreitol. Genetically, engineered variants of intactimmunoglobulin can be produced by obtaining a polynucleotide encodingthe antibody, and applying the general methods of molecular biology tosplice encoding sequences or introduce mutations and translate thevariant. Antibodies that are engineered variants of particular interestinclude chimeric and humanized antibodies, Fab-like fragments,single-chain variable region fragments (scFv), and diabodies.

Detectably Labeled Anti-Pregabalin Antibodies

The anti-pregabalin antibodies may also be labeled in order tofacilitate detection. A variety of protein labeling schemes may beemployed as desired depending on the intended use of the antibody, e.g.,immunoassay.

Examples of labels include labels that permit both the direct andindirect measurement of the presence of the antibody. Examples of labelsthat permit direct measurement of the antibody include radiolabels, suchas ³H or ¹²⁵I, fluorescent labels, dyes, microparticles, beads,chemiluminescent labels, colloidal particles, and the like. Examples oflabels which permit indirect measurement of the presence of the antibodyinclude enzymes where a substrate may provide for a colored orfluorescent product. For example, the antibodies may be labeled with acovalently bound enzyme capable of providing a detectable product signalafter addition of suitable substrate. Instead of covalently binding theenzyme to the antibody, the antibody may be modified to comprise a firstmember of specific binding pair which specifically binds with a secondmember of the specific binding pair that is conjugated to the enzyme,e.g., the antibody may be covalently bound to biotin and the enzymeconjugate to streptavidin. Examples of suitable enzymes for use inconjugates include horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like.

Immunoassays

The present disclosure provides immunoassay methods for assessing thepresence or absence of pregabalin in a sample of interest. Immunoassaysof the present disclosure can be of a variety of formats. Theimmunoassays may be separation immunoassays (also known as heterogeneousimmunoassays) or homogeneous immunoassays. Furthermore, the immunoassaysmay be qualitative or quantitative. Assays of this disclosure includeboth sandwich and competition assays. The immunoassays may embody assaysthat are neither sandwich nor competition assays, as in certain assaysinvolving immunoprecipitation.

In certain embodiments, the immunoassays of the present disclosure fordetecting the presence of absence of pregabalin in a sample can beconducted by adding, to a reaction mixture, (i) a sample suspected ofcontaining pregabalin and (ii) an anti-pregabalin antibody capable offorming of a complex with pregabalin that may be present in the sample;and detecting the presence or absence of the complex. The presence orabsence of the complex is indicative of the presence or absence ofpregabalin in the sample. Moreover, the amount of complex formed can beassessed to determine the concentration of pregabalin present in thesample (e.g., to provide an assessment of serum or tissue concentrationof pregabalin in a subject from whom the sample was obtained). Thepresence and/or amount of complex can be assessed directly (e.g., bydetecting bound antibody in the complex) or indirectly (e.g., byassessing activity of an enzyme in a pregabalin enzyme conjugate, wherewhen the pregabalin enzyme conjugate is not bound to antibody, adetectable signal is generated, indicating that the anti-pregabalinantibody in the reaction mixture has been bound by pregabalin from thesample).

In some embodiments, the immunoassays of the disclosure includecombining the sample with an anti-pregabalin antibody under conditionsthat permit the formation of a stable complex between the analyte to betested (e.g., pregabalin) and the antibody.

Assays may be performed in solution or may use a solid (insoluble)support (e.g. polystyrene, nitrocellulose, or beads), using any standardmethods (e.g., as described in Current Protocols in Immunology, Coliganet al., ed.; John Wiley & Sons, New York, 1992). Such methods includeELISAs (enzyme-linked immunosorbent assays), IRMAs (immunoradiometricassays), and RIAs (radioimmunoassays).

Where the assay is performed in solution, the test samples (and,optionally a control sample) may be incubated with an anti-pregabalinantibody for a time period sufficient to allow formation of analyte andaffinity reagent complexes, for example, from 1 min to 24 hrs, or more.As previously noted, the anti-pregabalin antibody may include adetectable label (e.g., radionuclide, fluorescer, or enzyme). The samplemay then be treated to separate the pregabalin-anti-pregabalin antibodycomplexes from excess, unreacted anti-pregabalin antibody (e.g., byaddition of an anti-anti-pregabalin antibody (e.g., anti-immunoglobulinantiserum) followed by centrifugation to precipitate the complexes, orby binding to an affinity surface such as a second, unlabeledanti-pregabalin antibody fixed to a solid substrate (such as Sepharose®or a plastic well). Detection of anti-pregabalin antibody bound to apregabalin may be achieved in a variety of ways. For example, ifnecessary, a substrate for the detectable label may be added to thesample.

Where the assay uses a solid support, the support can have ananti-pregabalin antibody (or pregabalin conjugate) bound to a supportsurface. Binding of the assay reagent may facilitate the stable,wash-resistant binding of pregabalin, which may be present in the sample(or anti-pregabalin antibody that is not bound to pregabalin from thesample, and is present in the reaction mixture, as in a competitivebinding assay) to the solid support via specific binding to theanti-pregabalin antibody. The insoluble supports may be any compositionto which antibodies or suitable pregabalin conjugates can be bound,which is readily separated from soluble material, and which is otherwisecompatible with the overall method of detection of anti-pregabalinantibody a sample.

The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which theanti-pregabalin antibody can be bound include beads, e.g. magneticbeads, membranes and microtiter plates. These can be composed of glass,plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose.

Assay reagents can include the anti-pregabalin antibodies as disclosedherein, as well as anti-anti-pregabalin antibodies, which may beoptionally detectably labeled. Methods for binding antibodies or otherproteins to solid supports may be used. After binding of an assayreagent to the support, the support may be treated with a blockingagent, which binds to the support in areas not occupied by the assayreagent. Suitable blocking agents include non-interfering proteins suchas bovine serum albumin, casein, gelatin, and the like. Alternatively,detergents at non-interfering concentrations, such as Tween, NP40,TX100, and the like may be used. Such blocking treatment may reducenonspecific binding.

Qualitative and Quantitative Methods

Assays of the present disclosure include both qualitative andquantitative assays. Typical quantitative methods involve mixing ananalyte with a pre-determined amount of the reagent antibody, andcorrelating the amount of complex formed with the amount of analyte inthe original sample. In some embodiments, the correlation is based on arelationship determined using standard samples containing known amountsof analyte in the range expected for the sample to be tested. In aqualitative assay, sufficient complex above or below a threshold levelestablished by samples known to contain or be free of analyte establishthe assay result. Unless otherwise stated, the act of “measuring” or“determining” in this disclosure refers to both qualitative andquantitative determination.

Samples

Samples may be biological samples taken from subjects suspected of beingadministered the analyte, e.g., pregabalin.

A sample suspected of containing or containing analyte refers to anysample which is reasonably suspected of containing analyte that can beanalyzed by the methods of the present disclosure. Such samples caninclude human, animal or man-made samples. The sample can be prepared inany convenient medium which does not interfere with the assay.Typically, the sample is an aqueous solution or a natural fluid, suchas, urine, whole blood, serum, plasma, cerebral-spinal fluid, or saliva.In some instances, the sample is serum or hair.

As used herein, a “sample” refers to a sample of tissue or fluidisolated from a subject, which in the context of the present disclosuregenerally refers to samples suspected of containing pregabalin, whichsamples, after optional processing, can be analyzed in an in vitroassay.

Examples of samples of interest include, but are not limited to, a“blood sample” (which as used herein is meant to include whole blood,plasma, serum, and the like), fecal matter, urine, tears, sweat saliva,milk, organs, biopsies, secretions of the intestinal and respiratorytracts, vitreous humor, and fluids obtainable during autopsy (such ascerebrospinal fluid). A “blood-derived sample” refers to a sample thatis prepared from blood or a fraction thereof, e.g., plasma or serum.Respiratory secretions (e.g., samples obtained from fluids or tissue ofnasal passages, lung, and the like), “Human serum”, as used herein,refers to the aqueous portion of human blood remaining after the fibrinand suspended material (such as cells) have been depleted.

Blood samples, such as serum samples, can be obtained by any suitablemethod. In some embodiments, a trough serum/plasma is used and theconcentration range is 12-20 mg. Sweat samples can be obtained using,for example, a PharmChek® sweat patch from Sudormed. The PharmChek®sweat patch includes a semi-occlusive dressing containing a medicalgrade cellulose blotter paper collection pad, covered by a thin layer ofpolyurethane and acrylate adhesives. At the end of the wear period, thepad is eluted with a suitable buffer, such as 2.5 mL of 0.2 M acetatebuffer with methanol at pH 5.0 (25:75) or with acetonitrile.Furthermore, the biological samples may also be tissue samples, whichare extracted into liquid medium for immunoassay. For example, hairsamples can be tested by extracting into a liquid medium. The samplesmay be diluted or modified to facilitate the assay.

The samples may be experimental samples generated by any chemical orbiological method. For example, the samples may be standards containingknown concentrations of pregabalin or other substances used for assaycalibration.

In some embodiments, the biological sample may be diluted in a suitablesolution prior to assaying. In some embodiments, a solution suitable fordiluting a biological sample will include a buffer, such as phosphatebuffered saline (PBS), and may include additional items, such as forexample, a non-specific blocking agent, such as bovine serum albumin(BSA), a detergent, such as Triton-X-100, and the like.

Where desired, appropriate control samples for the assay include blood,serum, or urine collected from human subjects who have not receivedpregabalin (i.e., a negative control), or samples which contain a known,predetermined amount of a pregabalin analyte (i.e., a positive control).Alternatively, test results can be compared to detectable signal levelsknown to be associated with the presence or absence of pregabalin and/orcorrelated with an amount of pregabalin, e.g., a serum level ofpregabalin.

The assays may optionally include use of a calibration standard.“Calibration standard”, as used herein, refers to an aqueous mediumcontaining pregabalin at a predetermined concentration. In someembodiments, a series of these calibration standards are available at aseries of predetermined concentrations. In some embodiments, thecalibration standard is stable at ambient temperature. In someembodiments, the calibration standards are in a synthetic matrix. Insome embodiments, the calibration standards are in a non-syntheticmatrix such as human serum.

In some embodiments, a suitable initial source for the human sample is ablood sample. As such, the sample employed in the subject assays isgenerally a blood-derived sample. The blood derived sample may bederived from whole blood or a fraction thereof, e.g., serum, plasma,etc., where in some embodiments the sample is derived from blood allowedto clot and the serum separated and collected to be used to assay.

In embodiments in which the sample is a serum or serum derived sample,the sample is generally a fluid sample. Any convenient methodology forproducing a fluid serum sample may be employed. In some embodiments, themethod employs drawing venous blood by skin puncture (e.g., fingerstick, venipuncture) into a clotting or serum separator tube, allowingthe blood to clot, and centrifuging the serum away from the clottedblood. The serum may then be collected and stored until assayed. Oncethe patient derived sample is obtained, the sample may be assayed todetermine the level of pregabalin analyte.

Immunoassay Reagents

Immunoassay reagents that find use alone or in combination in the assaysdescribed herein include anti-pregabalin antibodies, pregabalinconjugates, and pregabalin (e.g., as a control or in competitive bindingassays). Immunoassay reagents can be provided in a buffered aqueoussolution. Such solutions may include additional components such assurface active additives, organic solvents, defoamers, buffers,surfactants, and anti-microbial agents. Surface active additives areintroduced to maintain hydrophobic or low-solubility compounds insolution, and stabilize components in the solution. Examples includebulking agents such as betalactoglobulin (BLG) or polyethyleneglycol(PEG); defoamers and surfactants such as Tween-20, Plurafac A38, TritonX-100, Pluronic 25R2, rabbit serum albumin (RSA), bovine serum albumin(BSA), and carbohydrates. Examples of organic solvents can includemethanol and other alcohols. Various buffers may be used to maintain thepH of the solution during storage. Illustrative buffers include HEPES,borate, phosphate, carbonate, tris, barbital and the like.Anti-microbial agents also extend the storage life of the immunoassayreagent.

Anti-Pregabalin Antibodies

Immunoassays generally involve at least one anti-pregabalin antibody,which may be produced by the methods disclosed herein. In someembodiments, the assays involve using an antibody raised against apregabalin derivative-protein conjugate, such as an antibody having alow cross-reactivity with non-pregabalin molecules that may be presentin a reaction mixture. Anti-pregabalin antibodies can be polyclonal ormonoclonal antibodies, capable of specifically binding pregabalin.

Depending upon the assay format, the anti-pregabalin antibody can beoptionally detectably labeled, may be used in conjunction with asecondary antibody (e.g., an antibody that specifically binds ananti-pregabalin antibody) that may be detectably labeled. Examples ofdetectable labels for antibodies are described herein.

Pregabalin Conjugates

Pregabalin conjugates of the present disclosure find use as immunoassayreagents depending on the assay format. For example, a pregabalinconjugate can act as a competitive binding reagent in competitivebinding assays, or can provide for a detectable signal when not bound byan anti-pregabalin antibody (e.g., where the pregabalin conjugate is apregabalin G6PDH conjugate). Examples of pregabalin conjugates useful asimmunoassay reagents are described below.

Detectable Labels

A variety of detectable labels can be used in connection with thepregabalin conjugate assay reagents for use in the methods disclosedherein. Such detectable labels can be isotopic labels. In otherembodiments, the detectable labels are non-isotopic signal-generatingmoieties, such as fluorophores and enzymes. Exemplary detectable labelsare described below. It will be apparent that while the detectablelabels are described below in the context of their use in pregabalinconjugates, many can also be adapted for use with anti-pregabalinantibodies.

Fluorophores

“Fluorophore” as used herein refers to a moiety that itself fluoresces,can be made to fluoresce, or can provide for quenching of fluorescenceof a fluorophor of a FRET pair (e.g., as in a FRET pair). In principle,any fluorophore can be used in the assays of this invention. In general,the fluorophore is selected so as to be compatible for use in the assayformat desired, and selected so as to be relatively insensitive to theassay conditions, e.g., pH, polarity, temperature and ionic strength.

Examples of fluorophores can be characterized as having one or more ofthe following characteristics: (a) A fluorescence lifetime of greaterthan about 15 nsec; (b) An excitation wavelength of greater than about350 nm; (c) A Stokes shift (a shift to lower wave-length of the emissionrelative to absorption) of greater than about 20 nm; (d) For homogeneousassays described below, fluorescence lifetime should vary with bindingstatus; and (e) The absorptivity and quantum yield of the fluorophoreshould be high. The longer lifetime may be advantageous because it mayfacilitate measurement and may be more easily distinguishable from theRaleigh scattering (background). Excitation wavelengths greater than 350nm may facilitate a reduction in background interference because mostfluorescent substances responsible for background fluorescence inbiological samples are excited below 350 nm. A greater Stokes shift mayalso allow for less background interference.

The fluorophores generally have a functional group available forconjugation either directly or indirectly to a pregabalin intermediateto generate a pregabalin conjugate having the attached fluorophore.

Fluorophores for use in heterogenous assays can be relativelyinsensitive to binding status. In contrast, fluorophores for use inhomogeneous assay can be sensitive to binding status, i.e., thefluorescence lifetime may be alterable by binding so that bound and freeforms can be distinguished.

Examples of fluorophores useful in the present disclosure include, butare not limited to, naphthalene derivatives (e.g. dansyl chloride),anthracene derivatives (e.g. N-hydroxysuccinimide ester of anthracenepropionate), pyrene derivatives (e.g. N-hydroxysuccinimide ester ofpyrene butyrate), fluorescein derivatives (e.g. fluoresceinisothiocyanate), rhodamine derivatives (e.g. rhodamine isothiocyanate),phycoerythin, and Texas Red.

Enzymes

In some embodiments, the signal-generating moiety is an enzyme. Theenzyme can be selected so as to be stable to provide for desirableshelf-life, e.g., stable when stored for a period of three months ormore, or six months or more, at temperatures which are convenient tostore in the laboratory, normally −20° C., or above. The enzyme can beselected so as to have a satisfactory turnover rate at or near the pHoptimum for binding to the antibody, such as at about pH 6-10, or forexample from pH 6.0 to pH 8.0. A product of the enzymatic reactionfacilitated by the enzyme can be either formed or destroyed as a resultof the enzyme reaction, and can provide an enzyme reaction product whichabsorbs light in the ultraviolet region or the visible region, such as awavelength range of about 250-750 nm, for example from 300-600 nm. Theenzyme may also have a substrate (including cofactors) which has amolecular weight of 300 Da or more, or 500 Da or more. The enzyme whichis employed or other enzymes, with like activity, may not be present inthe sample to be measured, or can be easily removed or deactivated priorto the addition of the assay reagents. Also, the enzyme can be selectedso as to avoid the impact of any naturally occurring inhibitors for theenzyme that may be present in samples to be assayed or as some othercomponent of the reaction mixture.

Although enzymes of up to 600,000 molecular weight can be employed, insome embodiments, relatively low molecular weight enzymes will beemployed of from 10,000 to 300,000 molecular weight, such as from 10,000to 150,000 molecular weight, or from 10,000 to 100,000 molecular weight.Where an enzyme has a plurality of subunits the molecular weight refersto the enzyme and not to the subunits.

It may be desirable to select an enzyme that is susceptible todetectable labeling. In this instance, the enzyme can be detectablelabeled using appropriate detectable labels described herein.

Examples of enzymes include, but are not limited to: alkalinephosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphatedehydrogenase, lactate dehydrogenase, β-galactosidase, and urease. Agenetically engineered fragment of an enzyme may be used, such as thedonor and acceptor fragment of β-galactosidase utilized in CEDIAimmunoassays (see, e.g., Henderson D R et al. Clin Chem. 32(9):1637-1641(1986)); U.S. Pat. No. 4,708,929. These and other enzymes which can beused are described by Eva Engvall in Enzyme Immunoassay ELISA and EMITin Methods in Enzymology, 70:419-439 (1980) and in U.S. Pat. No.4,857,453.

In some embodiments, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH) and it is attached to a pregabalin derivative, thus forming apregabalin-reactive partner conjugate. An anti-pregabalin antibody usedin conjunction with such pregabalin conjugates can be selected so as tospecifically bind the pregabalin epitope presented by the pregabalinenzyme conjugate, and thus affect activity of the pregabalin enzymeconjugate.

For assays employing pregabalin-enzyme conjugates, as an example of aprotein conjugate, in which a hapten is labeled with an enzyme, thehapten can be attached to the enzyme by any suitable method. In certainembodiments, the chemistry described herein for formation of immunogenicprotein conjugates of pregabalin derivatives is also used to prepare theenzyme conjugate. In this way, the pregabalin moiety presented to theantibody can be similar to the pregabalin epitope to which the antibodyspecifically binds.

The selection procedure may include the use of a hapten-reactive partnerconjugate including G6PDH as the reactive partner and a pregabalinderivative as the hapten. The first step in selecting an antibody mayinclude testing the magnitude of antibody inhibition of ahapten-reactive partner conjugate. In this step, the goal is todetermine and select for those antibodies which significantly inhibitthe enzyme activity of G6PDH. Antibodies which perform well in the firsttest may then be subjected to a second test. Here, the antibody is firstincubated with pregabalin. Next the hapten-reactive partner conjugate isadded. An subject antibody preferentially binds to pregabalin instead ofthe pregabalin-reactive partner conjugate. The reduction in binding tothe hapten-reactive partner conjugate would be detectable as an increaseG6PDH activity.

Detection

Via Fluorescence

In certain embodiments, when a fluorescently labeled analyte (i.e.,pregabalin antigen or antibody) is employed, the fluorescence emitted isproportional (either directly or inversely) to the amount of analyte.The amount of fluorescence can be determined by the amplitude of thefluorescence decay curve for the fluorescent species. In some instances,this amplitude parameter is directly proportional to the amount offluorescent species, and accordingly to the analyte.

In some cases, spectroscopic measurement of fluorescence is accomplishedby: (a) exciting the fluorophore with a pulse of light; (b) detectingand storing an image of the excitation pulse and an image of all thefluorescence (the fluorescent transient) induced by the excitationpulse; (c) digitizing the image; (d) calculating the true fluorescenttransient from the digitized data; and (e) determining the amplitude ofthe fluorescent transient as an indication of the amount of fluorescentspecies.

According to the method, substantially all of the fluorescence emittedby the fluorescent species reaching the detector as a function of timefrom the instant of excitation is measured. As a consequence, the signalbeing detected is a superimposition of several component signals (forexample, background and one analyte specific signal). As mentioned, theindividual contributions to the overall fluorescence reaching thedetector can be distinguished based on the different fluorescence decayrates (lifetimes) of signal components. In order to quantitate themagnitude of each contribution, the detected signal data can beprocessed to obtain the amplitude of each component. In some instances,the amplitude of each component signal is proportional to theconcentration of the fluorescent species.

Via Enzyme

Detection of the amount of product produced by the hapten-reactivepartner conjugate of the invention can be accomplished by severalmethods. Among these methods are colorimetry, fluorescence, andspectrophotometry. These methods of detection are discussed in“Analytical Biochemistry” by David Holme, Addison-Wesley, 1998, which isincorporated herein by reference.

Solid Supports

The pregabalin conjugates and/or the anti-pregabalin antibodies to beused as reagents in an assay can be insolubilized by attachment to asolid phase. This can be, for example, a wall of a vessel containing thereagent, to a particulate, or to a large molecular weight carrier thatcan be kept in suspension but is removable by physicochemical means,such as centrifugation or microfiltration. The attachment need not becovalent, but is at least of sufficient permanence to withstand theseparation techniques (including washes) that may be part of the assayprocedure. Examples of particulate materials include, but are notlimited to, agarose, polystyrene, cellulose, polyacrylamide, latexparticles, magnetic particles, and fixed red cells. Examples ofcommercially available matrices include Sepharose® (Pharmacia), Poros®resins (Roche Molecular Biochemicals), Actigel Superflow™ resins(Sterogene Bioseparations Inc.), and Dynabeads™ (Dynal Inc.). In someinstances, the support used depends on such features as stability,capacity, accessibility of the coupled antibody, flow rate (or theability to disperse the resin in the reaction mixture), and ease ofseparation.

Assay Formats

As noted above, immunoassays for detection of pregabalin can be of avariety of formats. In certain embodiments, the immunoassays involvecombining one or more immunoassay reagents (e.g., at least ananti-pregabalin antibody) with a test sample (e.g., a sample suspectedof containing pregabalin) in a reaction mixture. “Reaction mixture”generally refers to the combination of a sample suspected of containingpregabalin and one or more immunoassay reagents as described in thepresent disclosure to facilitate detection of the presence or absence ofpregabalin in the sample, where the detection may be qualitative orquantitative. The reaction mixture is usually an aqueous solution,although the immunoassay reagent(s) may be in solution or immobilized ona support (e.g., a substrate such as a bead). The reaction mixture caninclude other components compatible with the immunoassay, e.g., buffers,and the like.

Immunoassays usually are classified in one of several ways. For example,immunoassays can be classified according to the mode of detection used,e.g., enzyme immunoassays, radio immunoassays, fluorescence polarizationimmunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.Another grouping is according to the assay procedure used, e.g.,competitive assay formats, sandwich-type assay formats as well as assaysbased on precipitation or agglutination principles. In some cases, afurther distinction is made depending on whether washing steps areincluded in the procedure (so-called heterogeneous assays) or whetherreaction and detection are performed without a washing step (so-calledhomogeneous assays). Examples of assays are described in more detailbelow.

Homogeneous and Heterogeneous Immunoassays

Immunoassays may be described as heterogeneous or homogeneous.“Homogeneous immunoassay”, as used herein, refers to an assay methodwhere the complex is typically not separated from unreacted reactioncomponents, but instead the presence of the complex is detected by aproperty which at least one of the reactants acquires or loses as aresult of being incorporated into the complex. Homogeneous assaysinclude systems involving fluorochrome and fluorochrome quenching pairson different reagents, enzyme and enzyme inhibitor pairs on differentreagents, chromophore and chromophore modifier pairs on differentreagents, and latex agglutination assays, and the like.

An example of a homogeneous assay is the quantitative homogeneous enzymeimmunoassay in which a pregabalin moiety is conjugated to an activeenzyme. In some instances, the conjugation is arranged so that thebinding of an anti-pregabalin antibody to the derivative affectsenzymatic activity in a qualitative or quantitative fashion. Forexample, if a sample containing pregabalin is premixed with theantibody, the antibody will complex with the pregabalin and be preventedfrom binding to the enzyme conjugate. In this way, the activity of theenzyme can be correlated with the amount of pregabalin present in thesample.

G6PDH is an example of an enzyme useful in such assays. In someembodiments, the G6PDH is a variant of a naturally-occurring G6PDH inwhich one or more lysine residues are deleted or substituted, or one ormore cysteine residues are introduced. For example, Leuconostocmesenteroides G6PDH are dimeric enzymes that have the ability tocatalyze the oxidation of D-glucose-6-phosphate toD-glucono-delta-lactone-6-phosphate by utilizing either NAD⁺ or NADP⁺.This property of using NAD⁺ differentiates these enzymes from humanG6PDH, which utilizes only NADP⁺ effectively, and allows L.mesenteroides-specific G6PDH activity to be measured in the presence ofhuman G6PDH, as for example in human samples. In some embodiments,G6PDHs from L. mesenteroides are used in homogeneous immunoassays. Twoexamples of genera of bacteria from which to select G6PDH areLeuconostoc and Zymomonas. Within these genera L. mesenteroides, L.citreum, L. lactis, L. dextranicum, and Z. mobilis may be used. Forexample, L. mesenteroides, L. citreum, L. lactis are specific examples.

FIG. 10 shows a scheme of a homogeneous, competitive immunoassay forpregabalin using an anti-pregabalin antibody and a pregabalin-G6PDHenzyme conjugate in a sample (e.g., a plasma sample). As shown inreaction scheme 1 in FIG. 10 , panel 1, in the absence of plasma drug(pregabalin), the anti-pregabalin antibody binds to thepregabalin-enzyme (G6PDH) conjugate and inactivates the enzyme. As shownin reaction scheme 2 in FIG. 10 , panel 2, in the presence of plasmadrug, pregabalin from plasma, if present, competes with thepregabalin-G6PDH conjugate for binding to the antibody, thus allowingsome fraction of the pregabalin-G6PDH conjugate to become active andconvert NAD⁺ to NADH. The active G6PDH produces an absorbance signalchange over time at 340 nm. In some instances, the analyte to bedetected (pregabalin) is exposed to the antibody before thepregabalin-G6PDH conjugate to minimize saturation of the antibody withpregabalin-G6PDH conjugate. In these embodiments, the assay includesincubation of the sample and antibody before addition of thepregabalin-G6PDH conjugate.

Another example of a homogeneous assay system is a cloned enzyme donorimmunoassay, described in more detail below.

In a separation-based or “heterogenous” assay, the detecting of acomplex of an anti-pregabalin antibody and an analyte may include aprocess where the complex formed is physically separated from eitherunreacted analyte, unreacted antibody, or both.

In certain embodiments of a heterogenous immunoassay, a complex of ananti-pregabalin antibody and an analyte may be first formed in the fluidphase, and then subsequently captured by a solid phase reagent orseparated on the basis of an altered physical or chemical property, suchas by gel filtration or precipitation. Alternatively, one of thereagents may be attached to a solid phase before contacting with otherreagents, and then the complex may be recovered by washing the solidphase free of unreacted reagents. Separation-based assays typicallyinvolve use of a labeled derivative or antibody to facilitate detectionor quantitation of the complex. Suitable labels include radioisotopessuch as ¹²⁵I, enzymes such as peroxidase and β-galactosidase, andfluorescent labels such as fluorescein isothiocyanate. In certaininstances, the separation step includes removing labeled reagent presentin complex form from unreacted labeled reagent. The amount of label inthe complex can be measured directly or inferred from the amount leftunreacted.

Sandwich and Competition Assays

Assays of the present disclosure include both sandwich and competitionassays. Sandwich assays typically involve forming a complex in which theanalyte to be measured is sandwiched between one reagent, such as afirst antibody used ultimately for separation of the complex, andanother reagent, such as a second antibody used as a marker for theseparated complex. Competition assays involve a system in which theanalyte to be measured competes with a derivative of the analyte forbinding to another reagent, such as an antibody. An example of acompetition assay using EMIT® is described in U.S. Pat. No. 3,817,837.

In some embodiments, the immunoassay includes adding a pregabalinconjugate having a pregabalin moiety and a detectable label to thesample. The presence or absence of pregabalin in the sample can bedetected by detecting the detectable label. The detectable label mayinclude an enzyme and the detecting may be performed by assaying theactivity of the enzyme. In some embodiments, the enzyme is adehydrogenase, such as G6PDH.

Lateral Flow Chromatography

Aspects of the present disclosure include lateral flow chromatography.In certain embodiments of lateral flow chromatography, a membrane stripthat includes a detection device, such as a non-isotopic signalgenerating moiety, is used for detecting pregabalin. A sample from apatient can be applied to the membrane strip. The sample interacts withthe detection device, producing a result. The results can signifyseveral things, including the absence of pregabalin in the sample, thepresence of pregabalin in the sample, and/or the concentration ofpregabalin in the sample.

In certain embodiments, a method of qualitatively determining thepresence or absence of pregabalin in a sample through the use of lateralflow chromatography is provided. In some embodiments, the qualitativelateral flow device includes: (1) A sample pad where the sample isapplied. The sample pad may be treated with chemicals such as buffers orsalts, which, when redissolved, facilitate reaction of the sample withthe conjugate, test, and control reagents; (2) A conjugate release pad,such as a polyester or glass fiber material that is treated with aconjugate reagent such as an antibody colloidal gold conjugate. Atypical process for treating a conjugate pad is to use impregnationfollowed by drying. In use, the liquid sample added to the test willredissolve the conjugate so that it will flow into the membrane; (3) Themembrane substrate is usually made of nitrocellulose or a similarmaterial whereby antibody capture components are immobilized; (4) Awicking pad can be used in tests where blood plasma is separated fromwhole blood. An impregnation process is usually used to treat this padwith reagents intended to condition the sample and promote cellseparation; (5) An absorbent pad can provide a reservoir for collectingfluids that have flowed through the device; and (6) The above layers andmembrane system can be laminated onto a plastic backing with adhesivematerial which serves as a structural member.

In certain embodiments, a method of qualitatively determining thepresence of a pregabalin in a sample through the use of lateral flowchromatography is provided. In some embodiments, the membrane stripincludes a sample pad, which is a conjugate release pad (CRP), whichincludes an antibody that is specific for the pregabalin. This antibodycan be conjugated to a non-isotopic signal-generating moiety, such as acolloidal gold particle. Other detection moieties useful in a lateralflow chromatography environment include dyes, colored latex particles,fluorescently labeled latex particles, non-isotopic signal generatingmoieties, etc. The membrane strip may include a capture line, in whichthe pregabalin derivative antigen is immobilized on the strip. In someembodiments, this immobilization is through covalent attachment to themembrane strip, optionally through a linker. In other embodiments, theimmobilization is through non-covalent attachment to the membrane strip.In still other embodiments, the immobile pregabalin derivative in thecapture line is attached to a reactive partner, such as an immunogeniccarrier like BSA.

Sample from a patient can be applied to the sample pad, where it cancombine with the antibody in the CRP, thus forming a solution. Thissolution may then migrate chromatographically by capillary action acrossthe membrane. When pregabalin is present in the sample, apregabalin-antibody complex can be formed, which migrates across themembrane by capillary action. When the solution reaches the captureline, the pregabalin-antibody complex may compete with the immobilepregabalin for the limited binding sites of the antibody. When asufficient concentration of pregabalin is present in the sample, it mayfill the limited antibody binding sites. This may prevent the formationof a colored antibody-immobile pregabalin complex in the capture line.Therefore, absence of color in the capture line indicates the presenceof pregabalin in the sample.

In the absence of pregabalin in the sample, a colored antibody-immobilepregabalin complex can form once the solution reaches the capture lineof the membrane strip. The formation of this complex in the capture lineis evidence of the absence of pregabalin therapeutic in the sample.

In certain embodiments, a method of quantitatively determining theamount of a pregabalin in a sample through the use of lateral flowchromatography is provided. Lateral flow chromatography is furtherdescribed in U.S. Pat. Nos. 4,391,904; 4,435,504; 4,959,324; 5,264,180;5,340,539; and 5,416,000, which are herein incorporated by reference. Insome embodiments, the antibody is immobilized along the entire length ofthe membrane strip. In certain cases, if the membrane strip is made frompaper, the antibody is covalently bound to the membrane strip. In somecases, if the membrane strip is made from nitrocellulose, then theantibody can be non-covalently attached to the membrane strip through,for example, hydrophobic and electrostatic interactions.

In certain embodiments, the membrane strip includes a CRP, whichincludes the pregabalin attached to a detector moiety. In someembodiments, the detector moiety is an enzyme, such as horseradishperoxidase (HRP).

Sample from a patient can be applied to the membrane strip, where it cancombine with the pregabalin/detector molecule in the CRP, thus forming asolution. This solution may then migrate chromatographically bycapillary action across the membrane. When pregabalin is present in thesample, both the sample pregabalin and the pregabalin/detector moleculecompete for the limited binding sites of the antibody. When a sufficientconcentration of pregabalin is present in the sample, it may fill thelimited antibody binding sites. This may cause the pregabalin/detectormolecule to continue to migrate in the membrane strip. The shorter thedistance of migration of the pregabalin/detector molecule in themembrane strip, the lower the concentration of pregabalin in the sample,and vice versa. When the pregabalin/detector molecule includes anenzyme, the length of migration of the pregabalin/detector molecule canbe detected by applying an enzyme substrate to the membrane strip.Detection of the product of the enzyme reaction may then be utilized todetermine the concentration of pregabalin in the sample. In someembodiments, the enzyme's color producing substrate, such as a modifiedN,N-dimethylaniline, is immobilized to the membrane strip and3-methyl-2-benzothiazolinone hydrazone is passively applied to themembrane, thus alleviating the need for a separate reagent to visualizethe color producing reaction.

Fluorescence Polarization Immunoassay for Pregabalin Fluorescencepolarization immunoassay (FPIA) technology is based on competitivebinding between an antigen/drug in a sample and a known concentration oflabeled antigen/drug. FPIA technology is described in, for example, U.S.Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and 4,751,190, which areincorporated herein by reference. Accordingly, the FPIA reagents,systems, and methods can be used with anti-pregabalin antibodies whichare also anti-pregabalin analog antibodies.

The FPIA technology can be used to identify the presence of pregabalinand can be used in assays that quantify the amount of pregabalin in asample. In part, the rotational properties of molecules in solutionallow for the degree of polarization to be directly proportional to thesize of the molecule. Accordingly, polarization increases as molecularsize increases. That is, when linearly polarized light is used to excitea fluorescent-labeled or other luminescent-labeled pregabalin orderivative thereof, which is small and rotates rapidly in solution, theemitted light is significantly depolarized. When the fluorescent-labeledpregabalin or derivative interacts with or is bound to an antibody, therotation is slowed and the emitted light is highly polarized. This isbecause the antibody significantly and measurably increases the size ofthe complex. Also, increasing the amount of unlabeled pregabalin in thesample can result in decreased binding of the fluorescent-labeledpregabalin or derivative by the anti-pregabalin antibody, and therebydecrease the polarization of light emitted from sample. The quantitativerelationship between polarization and concentration of the unlabeledpregabalin in the sample can be established by measuring thepolarization values of calibrations with known concentrations ofpregabalin. Thus, FPIA can be used to identify the presence andconcentration of pregabalin in a sample.

In some embodiments, the assay involves an FPIA assay system. An exampleof components of the FPIA system can include the following: (i)monoclonal or polyclonal anti-pregabalin antibodies capable ofspecifically binding to pregabalin and a pregabalin derivative; (ii) asample suspected of containing the pregabalin; and (iii) pregabalinderivative labeled with a fluorescent moiety, such as fluorescein. Insome cases, the system can be provided as a kit exclusive of the sample.Additionally, the system or kit can include various buffer compositions,pregabalin concentration gradient compositions or a stock composition ofpregabalin, and the like.

Homogeneous Microparticle Immunoassay for Pregabalin Homogeneousmicroparticles immunoassays (“HMI”), which can be referred to asimmunoturbidimetric assays, are based on the agglutination of particlesand compounds in solution. When particles and/or chemical compoundsagglutinate, particle sizes can increase and increase the turbidity of asolution. Accordingly, anti-pregabalin antibodies can be used withmicroparticles and pregabalin derivatives in order to assess thepresence, and optionally the amount, of pregabalin in a sample. HMItechnologies can be advantageous because the immunoassays can beperformed on blood, blood hemolysate, serum, plasma, tissue, and/orother samples. HMI assays can be configured to be performed withpregabalin and/or a pregabalin derivative loaded onto a microparticle,or with an anti-pregabalin antibody loaded onto a microparticle. HMI orimmunoturbidimetric assays can be used for measuring agglutination ofsubstances in a sample.

Immunoturbidimetric assay technologies are described in, e.g., U.S. Pat.Nos. 5,571,728, 4,847,209, 6,514,770, and 6,248,597, which areincorporated herein by reference. Such assays involve light attenuation,nephelometric, or turbidimetric methods. The formation of anagglutinated compound (AB) from pregabalin (A) and anti-pregabalinantibody microparticle binding partner (B) can be measured by the changewhich occurs in the scattering or absorption of the incident lightdirected into the sample. In some embodiments, the anti-pregabalinantibody (A) can bind with a pregabalin or derivative loadedmicroparticle. When suspendable particles having an immobilized bindingpartner are used, there can be an enhancement of the effects, whichmakes it possible to determine low pregabalin concentrations. Thesehomogeneous methods can be carried out quickly and simply, and permitthe automation of sample analyses as described in more detail below.

Cloned Enzyme Donor Immunoassays for Pregabalin

Cloned enzyme donor Immunoassays (“CEDIA”, Roche Diagnostics), are basedon the competition of pregabalin in the biological sample with apregabalin conjugate containing an inactive genetically engineeredenzyme-donor (“ED”) fragment, such as from β-D-galactosidegalactohydrolase or β-galactosidase (“β-gal”) from E. coli, for bindingto an antibody capable of binding pregabalin. If pregabalin is presentin the sample, it binds to the antibody, leaving the ED portion of theED-derivative conjugate free to restore enzyme activity ofβ-D-galactoside galactohydrolase or B gal in the reaction mixture so asto be capable of association with enzyme acceptor (“EA”) fragments. Theactive enzyme that includes the ED and EA is then capable of producing aquantifiable reaction product when exposed to an appropriate substrate.An example of a substrate is chlorophenol red-β-D-galactopyranoside(“CPRG”), which can be cleaved by the active enzyme into galactose andCPR, where CPR can be measured by absorbency at about wavelength 570 nm.When pregabalin is not present in the sample, the antibody binds to theED-derivative conjugate, thereby inhibiting association of the EDfragments with the EA fragments and inhibiting restoration of enzymeactivity. In some cases, the amount of reaction product and resultantabsorbance change are proportional to the amount of pregabalin in thesample.

Chemiluminescent Heterogeneous Immunoassays for Pregabalin

A competitive assay using chemiluminescent microparticle immunoassay(“CMIA”) technology can also be used to assess whether or not pregabalinis present in a sample. Various types of CMIA heterogeneous immunoassayscan be used for determining the presence and/or amount of a chemicalentity in a sample. CMIA assays can include the use of anti-pregabalinantibodies, which are capable of binding to pregabalin and itsderivatives, which are coupled to particles, such as magnetic particlesor particles suitable for separation by filtration, sedimentation,and/or other means. In some cases, a tracer, which can include apregabalin derivative linked to a suitable chemiluminescent moiety, canbe used to compete with free pregabalin in the patient's sample for thelimited amount of anti-pregabalin antibody on the particle. After thesample, tracer, and antibody particles interact, a wash step can be usedto remove unbound tracer. In certain instances, the amount of tracerbound to antibody particles can be measured by chemiluminescence, wherechemiluminescence is expressed in Relative Light Units (RULE). Theamount of chemiluminescence is inversely related to the amount of freedrug in the patient's sample and concentration is determined byconstructing a standard curve using known values of the drug.

Other Immunoassays for Pregabalin

The pregabalin derivatives, conjugates, antibodies, immunogens, and/orother conjugates described herein are also suitable for any of a numberof other heterogeneous immunoassays with a range of detection systemsincluding, but not limited to, enzymatic or fluorescent, and/orhomogeneous immunoassays including, but not limited to, rapid lateralflow assays, and antibody arrays. While various immunodiagnostic assayshave been described herein that utilize the pregabalin derivatives,conjugates, antibodies, immunogens and/or tracers, such assays can alsobe modified as desired. As such, various modifications of steps or actsfor performing such immunoassays can be made within the scope of thepresent disclosure.

Kits

The present disclosure also provides kits that find use in practicingthe subject methods, as described above. The kits of the presentinvention can include an anti-pregabalin antibody in a container, andmay include a pregabalin conjugate (e.g., for use in a competitivebinding assay, for use in an enzyme-based assay, and the like). The kitsmay also include a calibration standard and/or control standard usefulin performing the assay; and, optionally, instructions on the use of thekit. Kit components can be in a liquid reagent form, a lyophilized form,or attached to a solid support. The reagents may each be in separatecontainers, or various reagents can be combined in one or morecontainers depending on cross-reactivity and stability of the reagents.

The sample, suspected of containing a pregabalin, and a calibrationmaterial, containing a known concentration of the pregabalin, can beassayed under similar conditions. Pregabalin concentration may then becalculated by comparing the results obtained for the unknown specimenwith results obtained for the standard. In some cases, a calibration ordose response curve is used to compare the results obtained for theunknown specimen with results obtained for the standard.

Various ancillary materials may be employed in an assay in accordancewith the present disclosure. In certain embodiments, buffers and/orstabilizers are present in the kit components. In certain embodiments,the kits include indicator solutions or indicator “dipsticks”, blotters,culture media, cuvettes, and the like. In certain embodiments, the kitsinclude indicator cartridges (where a kit component is bound to a solidsupport) for use in an automated detector. In certain embodiments,additional proteins, such as albumin, or surfactants, particularlynon-ionic surfactants, may be included. In certain embodiments, the kitsinclude an instruction manual that describes an assay method asdisclosed herein and/or describes the use of the components of the kit.

Reagents and buffers used in the assays can be packaged separately or incombination into kit form to facilitate distribution. The reagents canbe provided in suitable containers, and can be provided in a packagealong with written instructions relating to assay procedures.

In certain embodiments, a kit for determining the presence or theabsence of pregabalin in a sample is provided. The kit may include ananti-pregabalin antibody and a pregabalin calibration standard. Thepregabalin calibration standard may include calibration and controlstandards useful in performing the assay. The kits can also optionallyinclude a conjugate of a pregabalin moiety and a detectable label. Incertain embodiments, a detectable label of the conjugate is an enzyme.In certain embodiments, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH). In some embodiments, the G6PDH is a variant of anaturally-occurring G6PDH in which one or more lysine residues aredeleted or substituted, or one or more cysteine residues are introduced.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use embodiments of the present disclosure, and are not intendedto limit the scope of what the inventors regard as their invention norare they intended to represent that the experiments below are all or theonly experiments performed. Also, it should be apparent that theembodiments can include additional embodiments not illustrated byexample. Additionally, many of the examples have been performed withexperimental protocols using the pregabalin derivatives, antigens,immunogens, and anti-pregabalin antibodies prepared in accordance withthe present disclosure. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Preparation of Amine Modified Pregabalin Derivative

Pregabalin (396 mg, 2.5 mmol) was taken into a 10% aqueous solution ofsodium carbonate (7 ml) and cooled in ice bath. A solution of Fmoc-Cl(647 mg, 2.5 mmol) in Dioxane (7 ml) was added drop wise. The reactionwas allowed to warm up to room temperature and stirred for 2 hrs. Water(50 ml) was added and extracted with Ethyl Acetate (2×50 ml). The waterlayer was cooled in ice bath and acidified to pH=1 with concentratedHCl, then extracted with ethyl acetate (3×50 ml). The combined organiclayer was washed with brine, dried over MgSO₄, and concentrated to give1.1 g of crude product, which was purified on silica gel using EthylAcetate in Hexane (0 to 30%) to give 800 mg (84%) of clean desiredintermediate 2.

Fmoc-pregabalin 2 (230 mg, 0.6 mmol) and DIEA (440 μl. 2.5 mmol) weremixed in dichloromethane (DCM; 10 ml) and added to 1 g of 2-Chlorotritylresin (1 mmol/g). After one hour of shaking, methanol (2 ml) was addedand the mix was shaken for 20 more minutes. Resin was drained, washedsuccessively with DCM and DMF (3×10 ml each).

A solution of Piperidine in DMF (30%, 10 ml) was added to the resin andshaken for 30 minutes. The reactions was drained and washed as describedabove. The resin was dried under vacuum and used for the next step.

Fmoc-glycine (892 mg, 3 mmol) and HATU (1.14 g, 3 mmol) were added to amixture of resin 3 in DMF (10 ml), DIEA (1.0 ml) was added and shakenfor 4 hrs. This reaction was repeated twice. The resin was drained andwashed with DMF, DCM and MeOH (3×30 ml each). A solution ofPiperidine/DMF (30%, 10 ml) was added and shaken for 30 minutes. Resinwas drained and washed as described above and dried under vacuum.

To a solution of 2-bromoacetic acid (556 mg, 4 mmol) in dry DMF (10 ml)was added DIC (6261, 4 mmol) and added to resin 4 (1.1 g). The mixturewas shaken for 30 minutes. This reaction was repeated twice. Resin wasdrained and washed with DMF, DCM, and methanol (3×20 ml each). The resinwas then dried under vacuum. The resin was taken in DCM (5 ml) and TFA(5 ml) was added and shaken for 1 hr. The resin was drained and washedwith 20% TFA/DCM solution (10 ml). The combined drained and washsolution was evaporated to dryness and triturated with ether. The crudeproduct was purified on silica gel using Methanol in DCM (0 to 10%) togive 115 mg of Hapten-A.

Mass; m/e=361.0 (100%), 363.0 (100%)

¹H NMR; 0.90 (d, 3H, J=6.4 Hz). 0.93 (d, 3H, J=6.4 Hz), 1.16 (m. 2H),1.66 (m. 1H), 1.69 (dd, 1H, J=9.6, J=15.2 Hz), 2.25 (m, 1H), 2.39 (dd,1H, J=2.4 Hz, J=15.2 Hz), 3.17 (m. 1H), 3.38 (m, 1H), 3.93 (s, 2H), 3.98(t, 2H, J=5.6 Hz), 7.02 (1s, 1H), 7.45 (1s, 1H).

Example 2 Preparation of Carboxylic Acid Modified Pregabalin Derivative

Pregabalin (2 g, 12.5 mmol) was dissolved in a mixture of water (5 ml)and dioxane (10 ml) then 1N NaOH (2 ml) was added and cooled to 5° C.(ice bath). Bocanhydride (3 g, 14 mmol) was then added and stirred onehour in an ice bath. The reaction mixture was allowed to warm up to roomtemperature and stirred fore 16 hrs. Dioxane was evaporated undervacuum, Water (50 ml) was added and cooled in ice bath and acidified topH=2-3. Extracted with ethyl acetate (3×50 ml). Organic layer was washedwith water (30 ml), dried over MgSO4 and concentrated to give 3.1 gdesired intermediate 5.

Intermediate 5 (200 mg, 0.77 mmol) was dissolved in DCM (10 ml). DIC(145 μl, 0.92 mmol) and HOBt (125 mg, 0.92 mmol) were added. After 5min, at room temperature, Alloc-1,2-diaminoethane 1.2 (134 mg, 0.92mmol) was added. After stirring for 16 hrs, solvent was evaporated,water (20 ml) was added and extracted with ethyl acetate (3×15 ml),washed with brine (15 ml) and dried over MgSO₄. After evaporation ofsolvent, 404 mg crude product was obtained, which was purified on silicagel using Methanol in DCM (0 to 5%) to give 200 mg (67%) of desiredproduct 6.

Intermediate 6 (194 mg, 0.50 mmol) was dissolved in DCM (10 ml).Dichlorobis(triphenylphosphine) palladium(II) (57 mg, 0.08 mmol) wasadded followed by Tributyltinhydride (200 μl, 0.75 mmol) and acetic acid(143 μl, 2.5 mmol). After overnight stirring, more palladium catalyst(42 mg, 0.05 mmol) was added and stirred for 4 hrs. No starting materialwas detected by TLC. Solvent was evaporated and the crude product wastriturated in ether (2×20 ml) and decanted and the oily crude was leftunder high vacuum for 2 hrs. This material was then dissolved in DCM (10ml) and hexane (20 ml) was added and left at 4° C. overnight. Solventwas decanted and the solid colorless precipitate left under vacuum togive 100 mg desired intermediate 7 pure enough to be used in the nextstep.

Intermediate 7 (100 mg, 0.33 mmol) in DCM (7 ml) was cooled in an icebath and Triethyl amine (50 μl, 0.35 mmol) was added followed byBromoacethyl bromide (67 μl, 0.33 mmol). The reaction mixture wasstirred in ice bath for 20 minutes until no starting material left(monitored by TLC). Solvent was removed under vacuum, water (5 ml) wasadded and extracted with ethyl acetate (2×10 ml). Organic layer wasdried over MgSO4 and concentrated to give 160 mg crude product, whichwas purified on silica gel using Ethyl Acetate/Hexanes (30% to 100%) togive 68 mg (50%) intermediate 8.

To a solution of intermediate 8 in DCM (4.5 ml), was added TFA (0.5 ml)at room temperature and stirred for 2 hrs. TLC showed no startingmaterial. Solvent was evaporated under vacuum and the residue trituratedwith ether (10 ml). The residue was then left under vacuum to give 68 mgHapten-B as a sticky solid.

Mass: m/e 322 (100% and 324 (100%), (M+1)

¹HNMR CDCl3, δ: 0.90 (d, 3H, J=6.8 Hz), 0.91 (d, 3H, J=6.8 Hz), 1.20 (t,2H, J=7.6 Hz), 1.62 (m, 1H), 2.23 (m, 1H), 2.29 (dd, 1H, J=8.8 Hz,J=14.8 Hz), 2.39 (dd, 1H, J=4 Hz, J=14.8 Hz), 2.81 (dd, 1H, J=8 Hz,J=12.8 Hz), 3.00 (dd, 1H, J=3.2 Hz, J=12.8 Hz), 3.32 (m, 2H), 3.43 (m,2H), 3.83 (s, 2H).

Example 3 Conjugation: Preparation of Pregabalin-SH-KLH Immunogen (KLH-Aand KLH-B)

a) Preparation of Thiolated KLH (KLH-SH)

One vial of lyophilized KLH (Pierce, 21 mg) was reconstituted with 3 mLof phosphate buffer (0.1 M, 0.15 M NaCl, 1 mM EDTA, pH 8.0). The KLHsolution was transferred to a reaction vial. Immediately beforereaction, 6-8 mg of SATA (N-Succinimidyl-S-acetylthioacetate) wasdissolved in 0.5 mL DMSO (resulted in −55 mM solution). 30 μl of theSATA solution was combined with 3.0 mL of protein solution (7 mg/mL).The contents were mixed and reaction incubated at room temperature forat least 30 minutes. A Sephadex G-50 column was equilibrated with twocolumn volumes of buffer (0.1 M phosphate, 0.15 M NaCl, pH 7.2-7.5). Thereaction mixture was applied to column. Fraction (500 μL) was collectedimmediately. The fractions that contain protein were identified bymeasuring absorbance at 280 nm. Protein fractions were pooled to give 12mL. Deacylation to generate a sulfhydryl for use in cross-linking wasaccomplished by adding 1.2 mL deacetylation solution (0.5 MHydroxylamine, 25 mM EDTA in PBS, pH 7.2-7.5). Contents were mixed andreaction incubated for 2 hours at room temperature. Sephadex G-50desalting column was used to purify the sulfhydryl-modified protein fromthe hydroxylamine in the deacetylation solution. The pooled fractionswere concentrated to 2.6 mL (8 mg/mL) using Amicon concentrator. SeeFIG. 3 .

b) Conjugation Hapten-A with Thiolated KLH (KLH-SH)

Dithiothreitol (DTT, 1 mM) was added to thiolated KLH to ensurereduction of disulfide bonds. The solution was allowed to mix overnightat 4° C. 10.2 mg Hapten-A was dissolved in 0.2 mL DMF. PregabalinHapten-A DMF solution was added in 5 to 10 μL quantities to a solutionof thiolated KLH-SH. The reaction was continued overnight at 4° C. Thissolution was dialyzed against three changes (2.0 liter each) of HEPESbuffer (10 mM, pH 7.0, 1 mM EDTA). This procedure yielded immunogen(KLH-A). See FIG. 5 .

c) Conjugation Hapten-B with Thiolated KLH (KLH-SH)

Dithiothreitol (DTT, 1 mM) was added to thiolated KLH to ensurereduction of disulfide bonds. The solution was allowed to mix overnightat 4° C. 10.2 mg Hapten-B was dissolved in 0.2 mL DMF. PregabalinHapten-B DMF solution was added in 5 to 10 μL quantities to a solutionof thiolated KLH-SH. The reaction was continued overnight at 4° C. Thissolution was dialyzed against three changes (2.0 liter each) of HEPESbuffer (10 mM, pH 7.0, 1 mM EDTA). This procedure yielded immunogen(KLH-B). See FIG. 6 .

Example 4 Conjugation: Native G6PDH and Pregabalin Containing ReactiveSulfhydryl Groups

a) Preparation of Thiolated Native G6PDH (G6PDH-SH)

The N-succinimidyl S-acetylthioacetate (SATA) reagent described abovewas used to introduce protected sulfhydryls into native G6PDH. 21 mgG6PDH was dialyzed against phosphate buffer (0.1 M, 0.15 M NaCl, 1 mMEDTA, pH 8.0). The dialyzed G6PDH was transferred to a reaction vial.Immediately before reaction, 6-8 mg of SATA was dissolved in 0.5 mL ofDMSO (resulted in −55 mM solution). Combined was 3.0 mL of proteinsolution (7 mg/mL) with 30 μl of the SATA solution. The contents weremixed and reaction incubated at room temperature for at least 60minutes. A 15 mL Sephadex G-50 column was equilibrated with two columnvolumes of buffer (0.1 M phosphate, 0.15 M NaCl, pH 7.2-7.5). Thereaction mixture was applied to the column. 1 mL fractions werecollected. Fractions that contain protein were identified by measuringfor those peaks having absorbance at 280 nm. 9 mL sample volume wascollected (approximating 21 mg enzyme). The enzyme was dialyzed againsta bicarbonate buffer (100 mM, pH 9.0) to give free SH groups. See FIG. 4.

b) Conjugation Hapten-B with Thiolated Native G6PHD

The pH of the enzyme solution of (a) above was adjusted to 7.2 with 0.1MHCl. Pregabalin Hapten-B (11.8 mg) was dissolved in 200 μL DMF.Pregabalin Hapten-B DMF solution was added in 5 to 10 μL quantities to asolution of 21 mg thiolated G6PDH. The reaction was continued overnightat 4° C. This solution was dialyzed against three changes (2.0 litereach) of HEPES buffer (10 mM, pH 7.0, 1 mM EDTA). See FIG. 8 .

Pregabalin Hapten-B is used in this Example. However, this conjugationtechnique is generally applicable to all Pregabalin Haptens containingbromoacetamido—such as Hapten-A.

Example 5 Conjugation: rG6PDH Containing Reactive Sulfhydryl Groups

Conjugation rG6PDH-SH was buffer exchanged with 50 mM phosphate-1.0 mMEDTA, pH 7.25. A solution of the protein (2 mL at 5 mg/mL) was thenmixed with a dithioerythreitol (25 mM final concentration in thephosphate-EDTA buffer) and mixture incubated at 4° C. for 16 hours. Theprotein solution was then buffer exchanged with 50 mM phosphate, 1.0 mMEDTA, 5 mM DTT, pH 7.25. The protein solution (2 mL at 5 mg/mL) wasmixed with 40 fold molar excess of a DMF solution (0.05 mL) of Hapten-Aand reaction mixture was stirred gently at 4° C. for 16 to 24 hours.Excess Hapten-A was separated from the enzyme-hapten conjugate bypassing the reaction mixture over a column of Sephadex G 50 in 50 mMphosphate, pH 7.0. The column fractions containing the enzyme-haptenconjugate were pooled by measuring absorption at 280 nm. See FIG. 7 .

Pregabalin Hapten-A was used in this Example. However, this conjugationtechnique was generally applicable to all Pregabalin Haptens to allHaptens Containing Reactive Bromoacetamido-Groups. Hapten-B can be usedin this conjugation procedure. See FIG. 9 .

Example 6 Preparation of Polyclonal Antibodies Reactive to Pregabalin

Polyclonal sera from 24 live rabbit were prepared by injecting theanimal with immunogen KLH-A and a second set of 24 live rabbits wereprepared by injecting the animal with immunogen KLH-B.

This immunogenic formulation included 200 g of the immunogen for thefirst immunization and 100 μg for all subsequent immunizations.Regardless of immunogen amount, the formulation was diluted to 1 mL withsterile saline solution. This solution was then mixed thoroughly with 1mL of the appropriate adjuvant: Freund's Complete Adjuvant for the firstimmunization or Freund's Incomplete Adjuvant for subsequentimmunizations. The stable emulsion was subsequently injectedsubcutaneously with a 19×1½ needle into New Zealand white rabbits.Injections were made at 3-4 week intervals. Bleeds of the immunizedrabbits were taken from the central ear artery using a 19×1 needle.Blood was left to clot at 37° C. overnight, at which point the serum waspoured off and centrifuged. Finally, preservatives were added in orderto form the polyclonal antibody material. Rabbit polyclonal antibodiesto pregabalin produced by the above procedure were designated as#16203-16226. Rabbit polyclonal antibody #16209 (bleeds P7-19 werepooled) was used in examples below.

The pregabalin antibodies and enzyme conjugates may be employed inassays for the detection of pregabalin. Either of the immunogens(KLH-A), or (KLH-B) can be injected into a mouse, sheep or rabbit toraise antibody.

Rabbit polyclonal antibody #16209 (bleeds P7-19 were pooled) wasscreened for curve size, precision, and specificity. The obtainedantibody was added into the antibody diluent to prepare the AntibodyReagent. The Antibody Reagent included antibody as prepared above,buffer, stabilizers, preservatives, and the substrates for the enzymeconjugate NAD⁺ and glucose 6 phosphate. Enzyme Conjugate includingG6PDH-B was added into the conjugate diluent to prepare the enzymeconjugate reagent. The enzyme Conjugate Reagent included the conjugate,buffer, stabilizers and preservatives. Enzyme conjugate G6PDH-B was usedwith rabbit polyclonal antibody #16209 (bleeds P7-19 were pooled) inexamples below. This technique is generally applicable to producepolyclonal antibodies to pregabalin derivatives.

The pregabalin antibodies and enzyme conjugates may be advantageouslyused in a homogeneous assay format to detect pregabalin in samples. Ananalyzer (instrument) useful to set up the assay was the Siemens Viva-ESystem. Pregabalin containing sample is incubated with antibody reagentfollowed by the addition of the enzyme conjugate reagent. The enzymeconjugate activity decreases upon binding to the antibody. The enzymeconjugate, which is not bound to the antibody, catalyzes the oxidationof glucose 6-phosphate (G6P). The oxidation of G6P is coupled with thereduction of NAD⁺ to NADH, which can be measured at 340 nm. The changein the absorbance at 340 nm can be measured spectrophotometrically. Thepregabalin concentration in a specimen can be measured in terms of G6PDHactivity. The increase in the rate at 340 nm is due to the formation ofNADH and is proportional to the enzyme conjugate activity.

Using the above procedure, an assay calibration curve was generated(FIG. 11 ) using pregabalin spiked into pooled pregabalin-free humanurine samples. The assay rate increased with increasing theconcentration of free drug in the sample.

Example 7 Immunoassay

The pregabalin antibodies and enzyme conjugates may be employed inassays for the detection of the analyte. Either of the immunogens;KLH-A, or KLH-B, is injected into a mouse, rabbit or sheep to raiseantibody. As described above, antibodies may be screened, and evaluatedfor properties such as specificity, conjugate inhibition, curve size andcross-reactivity. The obtained antibody is spiked into the antibodydiluent to prepare the Antibody Reagent. The Antibody Reagent includesantibody as prepared above, buffer, stabilizers, preservatives, and thesubstrates for the enzyme conjugate NAD⁺ and glucose-6-phosphate. Enzymeconjugate rG6PDH-A, or rG6PDH-B1 or rG6PDH-B2 or a label proteinincluding alkaline phosphatase, β-galactosidase and horse radishperoxidase is spiked into the conjugate diluent to prepare the EnzymeConjugate Reagent. The Enzyme Conjugate Reagent includes the conjugate,buffer, stabilizers and preservatives.

The pregabalin antibodies prepared as described in EXAMPLE 6 and enzymeconjugates prepared as described in EXAMPLE 4 or EXAMPLE 5 may be usedin a homogeneous assay format to detect pregabalin in urine samples. Ananalyzer (instrument) useful to set up the assay is Siemens Viva-E. TheSiemens Viva-E® System is an automated biochemistry analyzer used bymedical and toxicology laboratories to process biological fluidspecimens, such as urine, cerebrospinal fluid, and most commonly, blood.As illustrated in FIG. 10 , pregabalin containing urine sample isincubated with Antibody Reagent followed by the addition of the EnzymeConjugate Reagent. The enzyme conjugate activity decreases upon bindingto the antibody. The enzyme conjugate, which is not bound to theantibody, catalyzes the oxidation of glucose-6-phosphate. The oxidationof glucose-6-phosphate is coupled with the reduction of NAD⁺ to NADH,which can be measured at 340 nm. The change in the absorbance at 340 nmcan be measured spectrophotometrically. The pregabalin concentration ina urine specimen can be measured in terms of G6PDH activity. Theincrease in the rate at 340 nm is due to the formation of NADH and isproportional to the enzyme conjugate activity. An assay curve can begenerated using pregabalin spiked into pooled human pregabalin-freeurine. The assay rate increases with increasing the concentration offree drug in the sample.

Example 8 Preparation Calibrators

Pooled pregabalin-free human urine samples were fortified with EDTA, andNaN₃ preservative. Pregabalin stock solution was obtained fromCerilliant Corporation, (P-066, Pregabalin 1.0 mg/mL). Pregabalin stocksolution was added to the aliquots of pooled pregabalin-free human urinein preparing a series of known concentrations of pregabalin calibratorsranging from 0 to 2000 ng/mL. Table 1 below shows the concentrations ofcalibrators prepared by the aforementioned procedure.

TABLE 1 Calibrator and Control Concentration Calibrator (ng/mL) 0 100500 1000 2000

Example 9 Assay Performance

Standard Curve

Antibody Reagent was prepared by adding antibody #10930 to antibodydiluent. The Antibody Reagent was assayed with Enzyme Conjugate Reagentprepared with rG6PDH-B2 enzyme conjugate. Calibration curves weregenerated on the Siemens Viva-E® System by assaying each level ofcalibrator in duplicate. Calibrators were prepared as described inEXAMPLE 8. An example of these calibrator rates is shown in Table 2 anda typical calibration plot is provided in FIG. 11 .

TABLE 2 Calibrator Reaction Rate Pregabalin Concentration Reaction Rate(mA/min) (ng/mL) Average of Duplicates 0 324.3 100 344.0 500 381.5 1000407.0 2000 432.5

Analytical Recovery

Pooled pregabalin-free human urine samples were fortified with EDTA, andNaN₃ preservative was aliquoted in 10 mL portions spiked with knownquantity of pregabalin stock solution that was obtained from CerilliantCorporation, as described in EXAMPLE 8. These samples were used toassess analytical recovery. Analytical recovery was evaluated using foursamples. Testing was performed on the Siemens Viva-E® System asdescribed in EXAMPLE 7. Two calibration curves were generated and 3replicates of each sample were assayed each time with two runs. Mean,percent recovery and coefficient of variance (% CV) was calculated foreach level. The coefficient of variation represents the ratio of thestandard deviation to the mean, and it is a useful statistic forcomparing the degree of variation from one data series to another. Thecoefficient of variation is reported as a percentage. Data are providedin Table 3.

TABLE 3 Analytical Recovery Data Summary Spiked Level Mean Recovery(ng/mL) (ng/mL) % CV (%) 200 197.9 2.4 99.0 400 378.3 3.5 94.6 800 778.62.8 97.3 1500 1361.2 4.1 90.7

Specificity of the Immunoassay

The 20 L-amino acids and gabapentin were tested due to their similarchemical structure to pregabalin.

The specificity of the immunoassay was evaluated by adding a compound topooled pregabalin-free human urine samples fortified with EDTA, and NaN₃preservative and testing the samples and determining the percentcross-reactivity. Separate stock solutions of each compound wereprepared in methanol. Stock solution was added to pregabalin-free humanurine to give the concentrations shown in Table 4. Testing was performedon the Siemens Viva-E© System as described in EXAMPLE 7. The percentagecross-reactivity was calculated for each compound tested. Results areshown in Table 4.

TABLE 4 Cross-Reactivity Compound Concentration (μg/mL) %Crossreactivity Gabapentin 200 0.019 L-Arginine 200 0.006 L-Asparagine200 0.004 L-Aspartic Acid 200 0.004 L-Cysteine 200 0.005 L-Glutamic Acid200 0.006 L-Glycine 200 0.005 L-Histidine 200 0.007 L-Isoleucine 2000.008 L-Leucine 200 0.038 L-Methionine 200 0.031 L-Phenylalanine 2000.006 L-Serine 200 0.005 L-Threonine 200 0.005 L-Tyrosine 200 0.004L-Alanine 200 0.004 L-Lysine 200 0.001 L-Proline 200 0.004 L-Valine 2000.006 L-Tryptophan 200 0.006 L-Glutamine 200 0.004 % Cross-reactivity =100 × (mean value Test − mean value Control) ÷ (Concentration CompoundTested)

The preceding merely illustrates the principles of embodiments of thepresent disclosure. It will be appreciated that those skilled in the artwill be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theembodiments of the present disclosure and are included within its spiritand scope. Furthermore, all examples and conditional language recitedherein are principally intended to aid the reader in understanding theprinciples of the embodiments and the concepts contributed by theinventors to furthering the art, and are to be construed as beingwithout limitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the present disclosure as well as specific examplesthereof, are intended to encompass both structural and functionalequivalents thereof. Additionally, it is intended that such equivalentsinclude both currently known equivalents and equivalents developed inthe future, i.e., any elements developed that perform the same function,regardless of structure.

What is claimed is:
 1. A method for detecting pregabalin, the methodcomprising: combining in a reaction mixture: (a) a sample suspected ofcontaining pregabalin; and (b) a conjugate comprising a compound ofFormula (A):

wherein: R¹ or R² is —X—W-L-Z, wherein when R¹ is —X—W-L-Z, then R² is—NH₂, when R² is —X—W-L-Z, then R¹ is —OH, and X is NH; W is selectedfrom a bond, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, and carbonyl; L is a bond or alinker; and Z is an enzyme; or a salt thereof, and detecting thepresence of an enzymatic reaction product of the conjugate, wherein anincrease in the enzymatic reaction product of the conjugate indicatesthe presence of pregabalin in the sample.
 2. The method of claim 1,wherein W is a bond.
 3. The method of claim 1, wherein W is an alkyl orsubstituted alkyl.
 4. The method of claim 1, wherein W is a carbonyl. 5.The method of claim 1, wherein the linking group comprises 1-15 carbonatoms and/or 0-6 heteroatoms.
 6. The method of claim 1, wherein thelinker is selected from the group consisting of: —(CH₂)_(n)C(O)—,—C(O)(CH₂)_(n)—, —C(O)(CH₂)_(n)NH—C(O)—,—C(O)(CH₂)_(m)NH—C(O)(CH₂)_(n)—, —(CH₂)_(n)SCH₂C(O)—,—(CH₂)_(m)SCH₂C(O)(CH₂)_(n)—, —(CH₂)_(m)C(O)NH(CH₂)_(n)—,—(CH₂)_(n)NH—C(O)—, —(CH₂)_(m)NH—C(O)(CH₂)_(n)—, —C(O)—(CH₂)_(n)—, and—(CH₂)_(n)—; wherein m and n are each independently selected from aninteger from 0 to
 10. 7. The method of claim 1, wherein the linker is—(CH₂)_(m)NH—C(O)(CH₂)_(n)—, wherein m is 2 and n is
 1. 8. The method ofclaim 1, wherein the enzyme is selected from the group consisting ofglucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase,β-galactosidase, and horse radish peroxidase.
 9. The method of claim 8,wherein the enzyme is G6PDH.
 10. The method of claim 9, wherein theG6PDH comprises at least one cysteine per subunit.
 11. The method ofclaim 10, wherein the cysteine is not native to a naturally-occurringG6PDH.